Pyrrolobenzodiazepines and conjugates thereof

ABSTRACT

A conjugate of formula (A): Wherein Y is selected from formulae A1 and A2: Z 1  is a C 1-3  alkylene group; Z 2  is a C 1-3  alkylene group; Q is: where Q X  is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue; L is a linker connected to a cell binding agent; CBA is the cell binding agent; and n is an integer between 0 and 48.

The present invention relates to pyrrolobenzodiazepines (PBDs), in particular pyrrolobenzodiazepines having a linker group connected to a cell binding agent.

BACKGROUND TO THE INVENTION

Pyrrolobenzodiazepines

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise and bond to specific sequences of DNA; the preferred sequence is PuGPu. The first PBD antitumour antibiotic, anthramycin, was discovered in 1965 (Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965); Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Since then, a number of naturally occurring PBDs have been reported, and over 10 synthetic routes have been developed to a variety of analogues (Thurston, et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. and Thurston, D. E., Chem. Rev. 2011 111 (4), 2815-2864). Family members include abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206 (1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem. Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758 (1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980)), neothramycins A and B (Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97 (1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704 (1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin (Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin (Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of the general structure:

They differ in the number, type and position of substituents, in both their aromatic A rings and pyrrolo C rings, and in the degree of saturation of the C ring. In the B-ring there is either an imine (N═C), a carbinolamine(NH—CH(OH)), or a carbinolamine methyl ether (NH—CH(OMe)) at the N10-C11 position which is the electrophilic centre responsible for alkylating DNA. All of the known natural products have an (S)-configuration at the chiral C11a position which provides them with a right-handed twist when viewed from the C ring towards the A ring. This gives them the appropriate three-dimensional shape for isohelicity with the minor groove of B-form DNA, leading to a snug fit at the binding site (Kohn, In Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986)). Their ability to form an adduct in the minor groove, enables them to interfere with DNA processing, hence their use as antitumour agents.

A particularly advantageous pyrrolobenzodiazepine compound is described by Gregson et al. (Chem. Commun. 1999, 797-798) as compound 1, and by Gregson et al. (J. Med. Chem. 2001, 44, 1161-1174) as compound 4a. This compound, also known as SJG-136, is shown below:

Other dimeric PBD compounds, such as those bearing C2 aryl substituents in WO 2005/085251, have been disclosed, an example being:

These compounds have been shown to be highly useful cytotoxic agents.

Antibody-Drug Conjugates

Antibody therapy has been established for the targeted treatment of patients with cancer, immunological and angiogenic disorders (Carter, P. (2006) Nature Reviews Immunology 6:343-357). The use of antibody-drug conjugates (ADC), i.e. immunoconjugates, for the local delivery of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumor cells in the treatment of cancer, targets delivery of the drug moiety to tumors, and intracellular accumulation therein, whereas systemic administration of these unconjugated drug agents may result in unacceptable levels of toxicity to normal cells as well as the tumor cells sought to be eliminated (Xie et al (2006) Expert. Opin. Biol. Ther. 6(3):281-291; Kovtun et al (2006) Cancer Res. 66(6):3214-3121; Law et al (2006) Cancer Res. 66(4):2328-2337; Wu et al (2005) Nature Biotech. 23(9):1137-1145; Lambert J. (2005) Current Opin. in Pharmacol. 5:543-549; Hamann P. (2005) Expert Opin. Ther. Patents 15(9):1087-1103; Payne, G. (2003) Cancer Cell 3:207-212; Trail et al (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614).

Maximal efficacy with minimal toxicity is sought thereby. Efforts to design and refine ADC have focused on the selectivity of monoclonal antibodies (mAbs) as well as drug mechanism of action, drug-linking, drug/antibody ratio (loading), and drug-releasing properties (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No. 7,723,485; WO2009/052249; McDonagh (2006) Protein Eng. Design & Sel. 19(7): 299-307; Doronina et al (2006) Bioconj. Chem. 17:114-124; Erickson et al (2006) Cancer Res. 66(8):1-8; Sanderson et al (2005) Clin. Cancer Res. 11:843-852; Jeffrey et al (2005) J. Med. Chem. 48:1344-1358; Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070). Drug moieties may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

PBDs in ADCs

Dimeric PBDs have been disclosed as the drugs in drug conjugates. For example, in WO 2011/130598, dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody, are disclosed where the linker group is attached to one of the available N10 positions, and are generally cleaved by action of an enzyme on the linker group.

By contrast, in WO 2011/130613 and WO 2011/130616, dimer PBD compounds having linker groups for connection to a cell binding agent, such as an antibody, are disclosed where the linker group is attached via an aromatic group at one of the C2 positions, and are generally cleaved by action of an enzyme on the linker group. Such antibody drug conjugates are also described in Flyagre, J., et al, Chem. Biol. Drug Des. 81: 113-121 (2013), which also describes other types of antibody drug conjugates.

A further approach is described in WO 2007/085930, wherein tomamycin-like dimers have a linker group for connection to a cell binding agent, such as an antibody, where the linker group is attached to the tether between the tomamycin units, and are generally cleaved by action of an enzyme on the linker group.

The present inventors have developed a novel approach to forming PBD conjugates with cell binding agents, and in particular PBD antibody conjugates.

SUMMARY OF THE INVENTION

In a general aspect the present invention provides a conjugate comprising a PBD compound with a linker for connecting to a cell binding agent, wherein the linker is attached in a cleavable manner to the C7 position of the one PBD units. The cell binding agent is preferably an antibody. The invention also provides the PBD compound with the linking unit attached, and intermediates for their synthesis.

In a first aspect, the present invention provides a conjugate of formula A:

and salts and solvates thereof, wherein: D represents either group D1 or D2:

the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R² is selected from the group consisting of: (ia) C₅₋₁₀ aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃ alkylene; (ib) C₁₋₅ saturated aliphatic alkyl; (ic) C₃₋₆ saturated cycloalkyl; (id)

-   -    wherein each of R³¹, R³² and R³³ are independently selected         from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and         cyclopropyl, where the total number of carbon atoms in the R²         group is no more than 5;         (ie)

-   -    wherein one of R^(35a) and R^(35b) is H and the other is         selected from: phenyl, which phenyl is optionally substituted by         a group selected from halo, methyl, methoxy; pyridyl; and         thiophenyl; and         (if)

-   -    where R³⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃         alkenyl; C₂₋₃ alkynyl; cyclopropyl; phenyl, which phenyl is         optionally substituted by a group selected from halo, methyl,         methoxy; pyridyl; and thiophenyl;         (ig) halo;         when there is a single bond present between C2 and C3,         R² is

-   -    where R^(36a) and R^(36b) are independently selected from H, F,         C₁₋₄ saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl         groups are optionally substituted by a group selected from C₁₋₄         alkyl amido and C₁₋₄ alkyl ester; or, when one of R^(16a) and         R^(16b) is H, the other is selected from nitrile and a C₁₋₄         alkyl ester;         R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR,         NH₂, NHR, NRR′, NO₂, SnMe₃ and halo;         either     -   (a) R¹⁰ is H, and R¹¹ is OH or OR^(A), where R^(A) is C₁₋₄         alkyl; or     -   (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the         nitrogen and carbon atoms to which they are bound; or     -   (c) R¹⁰ is H and R¹¹ is OSO_(z)M, where z is 2 or 3 and M is a         monovalent pharmaceutically acceptable cation; or     -   (d) R¹¹ is OH or OR^(A), where R^(A) is C₁₋₄ alkyl and R¹⁰ is         selected from:

-   -    where R^(Z) is selected from:         -   (z-i)

-   -   -   (z-ii) OC(═O)CH₃;         -   (z-iii) NO₂;         -   (z-iv) OMe;         -   (z-v) glucoronide;         -   (z-vi) —C(═O)—X₁—NHC(═O)X₂—NH—R^(ZC), where —C(═O)—X₁—NH—             and —C(═O)—X₂—NH— represent natural amino acid residues and             R^(ZC) is selected from Me, OMe, OCH₂CH₂OMe;             Y is selected from formulae A1 and A2:

-   -   Z¹ is a C₁₋₃ alkylene group;     -   Z² is a C₁₋₃ alkylene group;     -   Q is:

-   -    where Q^(X) is such that Q is an amino-acid residue, a         dipeptide residue or a tripeptide residue;     -   L is a linker connected to a cell binding agent;     -   CBA is the cell binding agent;     -   n is an integer between 0 and 48;         R and R′ are each independently selected from optionally         substituted C₁₋₁₂ alkyl,         C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups, and optionally in         relation to the group NRR′, R and R′ together with the nitrogen         atom to which they are attached form an optionally substituted         4-, 5-, 6- or 7-membered heterocyclic ring;         R⁸ is either:         (a) independently selected from H, R, OH, OR, SH, SR, NH₂, NHR,         NRR′, NO₂, SnMe₃ and halo; or         (b) of formula A*:

wherein: D′ represents either group D′1 or D2:

wherein the dotted line indicates the optional presence of a double bond between C2′ and C3′; R¹⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, NO₂, SnMe₃ and halo; R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g. benzene or pyridine, which rings are optionally substituted; and X and X′ are independently selected from O, S and N(H); and R²², R¹⁶, R¹⁹, R²⁰ and R²¹ are as defined for R², R⁶, R⁹, R¹⁰ and R¹¹ respectively.

When R⁸ is A*, the compound is of the formula A*A:

A second aspect of the present invention provides novel drug-linker compounds of formula (B):

and salts and solvates thereof, wherein: Y^(L) is selected from formulae B1 and B2:

G is a linker for connecting to a cell binding agent; and the remaining groups are as defined in the first aspect.

A third aspect of the present invention also provides compounds of formula (C), which may be used in the preparation of the drug-linkers and conjugates of the invention:

and salts and solvates thereof, Y^(C) is selected from formulae C1 and C2:

and the remaining groups are as defined in the first aspect.

A fourth aspect of the present invention provides the use of a compound of the first aspect of the invention in a method of medical treatment. The fourth aspect also provides a pharmaceutical composition comprising a compound of the first aspect, and a pharmaceutically acceptable excipient.

A fifth aspect of the present invention provides a compound of the first aspect of the invention or a pharmaceutical composition of the fourth aspect of the invention for use in a method of treatment of a proliferative disease. The fifth aspect also provides the use of a compound of the first aspect in a method of manufacture of a medicament for the treatment of a proliferative disease, and a method of treating a mammal having a proliferative disease, comprising administering an effective amount of a compound of the first aspect or a pharmaceutical composition of the fourth aspect.

A sixth aspect of the present invention provides a method of synthesis of a compound of the first aspect of the present invention, comprising the step of conjugating a drug-linker of the second aspect with a cell-binding agent.

A seventh aspect of the present invention provides a method of synthesis of a drug-link of the second aspect, comprising the step of reacting a compound of the third aspect with one or more suitable reagents.

DETAILED DESCRIPTION OF THE INVENTION

Preferences

The following preferences may apply to all aspects of the invention as described above, or may relate to a single aspect. The preferences may be combined together in any combination.

D

In some embodiments, D is D1.

In some embodiments, D is D2.

R⁸

In some embodiments, R⁸ may be independently selected from H, OH, OR, SH, SR, NH₂, NHR, NRR′, and halo.

In some embodiments, R⁸ may be independently selected from H, OH and OR, where R may be selected from optionally substituted C₁₋₇ alkyl, C₃₋₁₀ heterocyclyl and C₅₋₁₀ aryl groups. R in R⁸ may in some of these embodiments be a C₁₋₄ alkyl group, which may or may not be substituted. A substituent of interest is a C₅₋₆ aryl group (e.g. phenyl).

In some embodiments, R⁸ is selected from OMe and OCH₂Ph.

In some embodiments, R⁸ is of formula A*, such that the compound is a PBD dimer.

Dimer Link

X and X′ are preferably O.

R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g. benzene or pyridine, which rings are optionally substituted.

In some embodiments, R″ may be C₃₋₁₂ alkylene group, which chain may be interrupted by one or more heteroatoms and/or aromatic rings, e.g. benzene or pyridine.

In some embodiments, R″ may be C₃₋₁₂ alkylene group which is optionally interrupted by one or more heteroatoms selected from O, S, and NMe and/or aromatic rings, which rings are optionally substituted.

In some embodiments, the aromatic ring is a C₅₋₂₀ arylene group, where arylene pertains to a divalent moiety obtained by removing two hydrogen atoms from two aromatic ring atoms of an aromatic compound, which moiety has from 5 to 20 ring atoms. The ring substituents may include —NH₂, CO₂H, SO₃H and N-methylpiperazine.

In some embodiments, R″ may be a C₃₋₁₂ alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g. benzene or pyridine, which rings are optionally substituted by NH₂.

In some embodiments, R″ may be C₃₋₁₂ alkylene group.

In some embodiments, R″ may be selected from a C₃, C₅, C₇, C₉ and a C₁₁ alkylene group.

In some embodiments, R″ may be selected from a C₃, C₅ and a C₇ alkylene group.

In some embodiments, R″ may be selected from a C₃ and a C₅ alkylene group.

In some embodiments, R″ is a C₃ alkylene group.

In some embodiments, R″ is a C₅ alkylene group.

The alkylene groups listed above may be optionally interrupted by one or more heteroatoms and/or aromatic rings, e.g. benzene or pyridine, which rings are optionally substituted.

The alkylene groups listed above may be optionally interrupted by one or more heteroatoms and/or aromatic rings, e.g. benzene or pyridine.

The alkylene groups listed above may be unsubstituted linear aliphatic alkylene groups.

R″ is preferably a C₃₋₇ alkylene group with no substituents. More preferably R″ is a C₃, C₅ or C₇ alkylene. Most preferably, R″ is a C₃ or C₅ alkylene.

R⁶

In some embodiments, R⁶ may be independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, NO₂, SnMe₃ and halo.

In some embodiments, R⁶ may be independently selected from H, OH, OR, SH, NH₂, NO₂ and halo.

In some embodiments, R⁶ is independently selected from H and halo.

In some embodiments, R⁶ is independently H.

These embodiments also apply to R¹⁶.

R⁹

In some embodiments, R⁹ may be independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, NO₂, SnMe₃ and halo.

In some embodiments, R⁹ is independently H.

These embodiments also apply to R¹⁹.

R¹⁷

In some embodiments, R¹⁷ may be independently selected from H, OH, OR, SH, SR, NH₂, NHR, NRR′, and halo.

In some embodiments, R¹⁷ may be independently selected from H, OH and OR, where R may be selected from optionally substituted C₁₋₇ alkyl, C₃₋₁₀ heterocyclyl and C₅₋₁₀ aryl groups. R in R¹⁷ may in some of these embodiments be a C₁₋₄ alkyl group, which may or may not be substituted. A substituent of interest is a C₅₋₆ aryl group (e.g. phenyl).

In some embodiments, R¹⁷ is selected from OMe and OCH₂Ph.

R²

When R² is a C₅₋₁₀ aryl group, in some embodiments it may be a C₅₋₇ aryl group. A C₅₋₇ aryl group may be a phenyl group or a C₅₋₇ heteroaryl group, for example furanyl, thiophenyl and pyridyl. In some embodiments, R² may be phenyl. In other embodiments, R² may be thiophenyl, for example, thiophen-2-yl and thiophen-3-yl.

When R² is a C₅₋₁₀ aryl group, it some embodiments it may be a C₈₋₁₀ aryl, for example a quinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl group may be bound to the PBD core through any available ring position. For example, the quinolinyl may be quinolin-2-yl, quinolin-3-yl, quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl and quinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may be preferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl, isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yl and isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl may be preferred.

When R² is a C₅₋₁₀ aryl group, it may bear any number of substituent groups. In some embodiments, it may bear from 1 to 3 substituent groups. In some embodiments, it may bear 1 or 2 substituent groups. In some embodiments, it may bear a single substituent group. The substituents may be any position.

Where R² is C₅₋₇ aryl group, in some embodiments a single substituent may be on a ring atom that is not adjacent the bond to the remainder of the compound, i.e. it may be β or γ to the bond to the remainder of the compound. Therefore, in embodiments where the C₅₋₇ aryl group is phenyl, the substituent may be in the meta- or para-positions, or may be in the para-position.

Where R² is a C₈₋₁₀ aryl group, for example quinolinyl or isoquinolinyl, in some embodiments there may be any number of substituents at any position of the quinoline or isoquinoline rings. In some embodiments, it bears one, two or three substituents, and these may be on either the proximal and distal rings or both (if more than one substituent).

R² Substituents, when R² is a C₅₋₁₀ Aryl Group

In embodiments where a substituent on R² when R² is a C₅₋₁₀ aryl group is halo, it may be F or Cl, and in some of these embodiments Cl.

In embodiments where a substituent on R² when R² is a C₅₋₁₀ aryl group is ether, it may in some embodiments be an alkoxy group, for example, a C₁₋₇ alkoxy group (e.g. methoxy, ethoxy) or it may in some embodiments be a C₅₋₇ aryloxy group (e.g phenoxy, pyridyloxy, furanyloxy). The alkoxy group may itself be further substituted, for example by an amino group (e.g. dimethylamino).

In embodiments where a substituent on R² when R² is a C₅₋₁₀ aryl group is C₁₋₇ alkyl, it may be a C₁₋₄ alkyl group (e.g. methyl, ethyl, propryl, butyl).

In embodiments where a substituent on R² when R² is a C₅₋₁₀ aryl group is C₃₋₇ heterocyclyl, it may be C₆ nitrogen containing heterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl, piperazinyl. These groups may be bound to the rest of the PBD moiety via the nitrogen atom. These groups may be further substituted, for example, by C₁₋₄ alkyl groups. If the C₆ nitrogen containing heterocyclyl group is piperazinyl, the said further substituent may be on the second nitrogen ring atom.

In embodiments where a substituent on R² when R² is a C₅₋₁₀ aryl group is bis-oxy-C₁₋₃ alkylene, this may be bis-oxy-methylene or bis-oxy-ethylene.

In embodiments where a substituent on R² when R² is a C₅₋₁₀ aryl group is ester, this is preferably methyl ester or ethyl ester.

In some embodiments, substituents when R² is a C₅₋₁₀ aryl group may include methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino, methylthiophenyl, dimethylaminopropyloxy and carboxy.

In some embodiments, R² may be selected from 4-methoxy-phenyl, 3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl, 4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl, 4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl, isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl, methoxynaphthyl, naphthyl, 4-nitrophenyl, 4-(4-methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.

When R² is C₁₋₅ saturated aliphatic alkyl, it may be methyl, ethyl, propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl or propyl (n-pentyl or isopropyl). In some of these embodiments, it may be methyl. In other embodiments, it may be butyl or pentyl, which may be linear or branched.

When R² is C₃₋₆ saturated cycloalkyl, it may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.

When R² is

-   -    in some embodiments, the total number of carbon atoms in the R²         group is no more than 4 or no more than 3.

In some embodiments, one of R³¹, R³² and R³³ is H, with the other two groups being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

In other embodiments, two of R³¹, R³² and R³³ are H, with the other group being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

In some embodiments, the groups that are not H are selected from methyl and ethyl. In some of these embodiments, the groups that are not H are methyl.

In some embodiments, R³¹ is H.

In some embodiments, R³² is H.

In some embodiments, R³³ is H.

In some embodiments, R³¹ and R³² are H.

In some embodiments, R³¹ and R³³ are H.

In some embodiments, R³² and R³³ are H.

A R² group of particular interest is:

When R² is

-   -    in some embodiments, the group (R^(35a) or R^(35b)) which is         not H is optionally substituted phenyl. If the phenyl optional         substituent is halo, it may be fluoro. In some embodiment, the         phenyl group is unsubstituted.

When R² is

-   -    in some embodiments where R³⁴ is phenyl, it is unsubstituted.         In other embodiments, the phenyl group bears a single fluoro         substituent. In other embodiments, R¹⁴ is selected from H,         methyl, ethyl, ethenyl and ethynyl. In some of these         embodiments, R¹⁴ is selected from H and methyl.

When R² is halo, in some embodiments, it is fluoro.

When there is a single bond present between C2 and C3, R² is

In some embodiments, R^(36a) and R^(36b) are both H.

In other embodiments, R^(36a) and R^(36b) are both methyl.

In further embodiments, one of R^(36a) and R^(36b) is H, and the other is selected from C₁₋₄ saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups are optionally substituted. In some of these further embodiment, the group which is not H may be selected from methyl and ethyl.

R²²

The above preferences for R² when there is a double bond present between C2 and C3 apply equally to R²², when there is a double bond present between C2′ and C3′.

The above preferences for R² when there is a single bond present between C2 and C3 apply equally to R²², when there is a single bond present between C2′ and C3′.

N10-C11

In some embodiment, R¹⁰ is H, and R¹¹ is OH, OR^(A), where R^(A) is C₁₋₄ alkyl. In some of these embodiments, R¹¹ is OH. In others of these embodiments, R¹¹ is OR^(A), where R^(A) is C₁₋₄ alkyl. In some of these embodiments, R^(A) is methyl.

In some embodiments, R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.

In some embodiments, R¹⁰ is H and R¹¹ is OSO_(z)M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation. In some of these embodiments, M is a monovalent pharmaceutically acceptable cation, and may be Na⁺. Furthermore, in some embodiments z is 3.

In some embodiments where R¹⁰ is (d-iii), there may be an additional nitro group on the benzene ring, e.g. ortho to R^(Z).

In some embodiments, R¹¹ is OH or OR^(A), where R^(A) is C₁₋₄ alkyl and R¹⁰ is selected from:

—C(═O)—X₁—NHC(═O)X₂—NH— represent a dipeptide. The amino acids in the dipeptide may be any combination of natural amino acids. The dipeptide may be the site of action for cathepsin-mediated cleavage.

In one embodiment, the dipeptide, —C(═O)—X₁—NHC(═O)X₂—NH—, is selected from:

-   -   -Phe-Lys-,     -   -Val-Ala-,     -   -Val-Lys-,     -   -Ala-Lys-,     -   -Val-Cit-,     -   -Phe-Cit-,     -   -Leu-Cit-,     -   -Ile-Cit-,     -   -Phe-Arg-,     -   -Trp-Cit-,         where Cit is citrulline.

Preferably, the dipeptide, —C(═O)—X₁—NHC(═O)X₂—NH—, is selected from:

-   -   -Phe-Lys-,     -   -Val-Ala-,     -   -Val-Lys-,     -   -Ala-Lys-,     -   -Val-Cit-.

Most preferably, the dipeptide, —C(═O)—X₁—NHC(═O)X₂—NH—, is -Phe-Lys- or -Val-Ala-.

Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference.

In one embodiment, the amino acid side chain is derivatised, where appropriate. For example, an amino group or carboxy group of an amino acid side chain may be derivatised.

In one embodiment, an amino group NH₂ of a side chain amino acid, such as lysine, is a derivatised form selected from the group consisting of NHR and NRR′.

In one embodiment, a carboxy group COOH of a side chain amino acid, such as aspartic acid, is a derivatised form selected from the group consisting of COOR, CONH₂, CONHR and CONRR′.

In one embodiment, the amino acid side chain is chemically protected, where appropriate.

The side chain protecting group may be a group as discussed above. The present inventors have established that protected amino acid sequences are cleavable by enzymes. For example, it has been established that a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog. Additional protecting group strategies are set out in Protective Groups in Organic Synthesis, Greene and Wuts.

Possible side chain protecting groups are shown below for those amino acids having reactive side chain functionality:

-   -   Arg: Z, Mtr, Tos;     -   Asn: Trt, Xan;     -   Asp: Bzl, t-Bu;     -   Cys: Acm, Bzl, Bzl-OMe, Bzl-Me, Trt;     -   Glu: Bzl, t-Bu;     -   Gln: Trt, Xan;     -   His: Boc, Dnp, Tos, Trt;     -   Lys: Boc, Z—Cl, Fmoc, Z, Alloc;     -   Ser: Bzl, TBDMS, TBDPS;     -   Thr: Bz;     -   Trp: Boc;     -   Tyr: Bzl, Z, Z—Br.

In one embodiment, the side chain protection is selected to be orthogonal to a group provided as, or as part of, a capping group, where present. Thus, the removal of the side chain protecting group does not remove the capping group, or any protecting group functionality that is part of the capping group.

In other embodiments of the invention, the amino acids selected are those having no reactive side chain functionality. For example, the amino acids may be selected from: Ala, Gly, lie, Leu, Met, Phe, Pro, and Val.

It is particularly preferred in the present invention, that if L¹ comprises a dipeptide, then —C(═O)—X₁—NHC(═O)X₂—NH— is the same dipeptide.

Other preferred R¹⁰ groups include:

The above preferences apply equally to R²⁰ and R²¹.

R and R′

In some embodiments, R is independently selected from optionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups. These groups are each defined in the substituents section below.

In some embodiments, R is independently optionally substituted C₁₋₁₂ alkyl. In other embodiments, R is independently optionally substituted C₃₋₂₀ heterocyclyl. In further embodiments, R is independently optionally substituted C₅₋₂₀ aryl. In further embodiments, R is independently optionally substituted C₁₋₁₂ alkyl.

Described above in relation to R² are various embodiments relating to preferred alkyl and aryl groups and the identity and number of optional substituents. The preferences set out for R² as it applies to R are applicable, where appropriate, to all other groups R.

The preferences for R apply also to R′.

In some embodiments of the invention there is provided a compound having a substituent group —NRR′. In one embodiment, R and R′ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring. The ring may contain a further heteroatom, for example N, O or S. In some of these embodiments, the heterocyclic ring is itself substituted with a group R. Where a further N heteroatom is present, the substituent may be on the N heteroatom.

Dimers

In some embodiments, the groups X′, D, R¹⁶, R¹⁹, R²⁰ and R²¹ are the same as the groups X, D′, R⁶, R⁹, R¹⁰ and R¹¹ respectively. In these embodiments, the PBD monomer units have the same substituents except for at the 7 position.

Particularly preferred compounds of the first aspect of the present invention may be of formula Ia:

where R¹⁰, R¹¹, R²⁰, R²¹ and Y are as defined above; m is 1 or 3; R^(1a) is methyl or phenyl; and R^(2a) is selected from:

Particularly preferred compounds of the first aspect of the present invention may be of formula Ib:

where R¹⁰, R¹¹, R²⁰, R²¹ and Y are as defined above; m is 1 or 3; and R^(1a) is methyl or phenyl.

Particularly preferred compounds of the second aspect of the present invention may be of formula IIa:

where R¹⁰, R¹¹, R²⁰, R²¹ and Y^(L) are as defined above; m is 1 or 3; R^(1a) is methyl or phenyl; and R^(2a) is selected from:

Particularly preferred compounds of the second aspect of the present invention may be of formula IIb:

where R¹⁰, R¹¹, R²⁰, R²¹ and Y^(L) are as defined above; m is 1 or 3; and R^(1a) is methyl or phenyl.

Particularly preferred compounds of the third aspect of the present invention may be of formula IIIa:

where R¹⁰, R¹¹, R²⁰, R²¹ and Y^(C) are as defined above; m is 1 or 3; R^(1a) is methyl or phenyl; and R^(2a) is selected from:

Particularly preferred compounds of the third aspect of the present invention may be of formula IIIb:

where R¹⁰, R¹¹, R²⁰, R²¹ and Y^(C) are as defined above; m is 1 or 3; and R^(1a) is methyl or phenyl. Z¹, Z², Z³

In some embodiments, Z¹ is methylene. In some embodiments, Z¹ is ethylene. In some embodiments, Z¹ is propylene.

In some embodiments, Z² is methylene. In some embodiments, Z² is ethylene. In some embodiments, Z² is propylene.

In some embodiments, Z³ is methylene. In some embodiments, Z³ is ethylene. In some embodiments, Z³ is propylene.

n (Y, Y^(L))

In some embodiments, n (in Y or Y^(L)) is an integer between 0 and 24.

In some embodiments, n (in Y or Y^(L)) is an integer between 0 and 12.

In some embodiments, n (in Y or Y^(L)) is an integer between 0 and 8.

In some embodiments, n (in Y or Y^(L)) is an integer between 0 and 6.

In some embodiments, n (in Y or Y^(L)) is 0.

In some embodiments, n (in Y or Y^(L)) is 1.

In some embodiments, n (in Y or Y^(L)) is 2.

In some embodiments, n (in Y or Y^(L)) is 3.

In some embodiments, n (in Y or Y^(L)) is 4.

In some embodiments, n (in Y or Y^(L)) is 5.

In some embodiments, n (in Y or Y^(L)) is 6.

In some embodiments, n (in Y or Y^(L)) is 7.

In some embodiments, n (in Y or Y^(L)) is 8.

In some embodiments, Z¹ is methylene and n is 3.

In some embodiments, Z² is propylene and n is 8.

Q^(X)

In one embodiment, Q is an amino acid residue. The amino acid may a natural amino acids or a non-natural amino acid.

In one embodiment, Q is selected from: Phe, Lys, Val, Ala, Cit, Leu, lie, Arg, and Trp, where Cit is citrulline.

In one embodiment, Q comprises a dipeptide residue. The amino acids in the dipeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the dipeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the dipeptide is the site of action for cathepsin-mediated cleavage. The dipeptide then is a recognition site for cathepsin.

In one embodiment, Q is selected from:

-   -   ^(NH)-Phe-Lys-^(C═O),     -   ^(NH)Val-Ala-^(C═O),     -   ^(NH)Val-Lys-^(C═O),     -   ^(NH)Ala-Lys-^(C═O),     -   ^(NH)-Val-Cit-^(C═O),     -   ^(NH)-Phe-Cit-^(C═O),     -   ^(NH)-Leu-Cit-^(C═O),     -   ^(NH)-Ile-Cit-^(C═O),     -   ^(NH)-Phe-Arg-^(C═O), and     -   ^(NH)-Trp-Cit-^(C═O);         where Cit is citrulline.

Preferably, Q is selected from:

-   -   ^(NH)-Phe-Lys-^(C═O),     -   ^(NH)-Val-Ala-^(C═O),     -   ^(NH)-Val-Lys-^(C═O),     -   ^(NH)-Ala-Lys-^(C═O), and     -   ^(NH)-Val-Cit-^(C═O).

Most preferably, Q is selected from ^(NH)-Phe-Lys-^(C═O), ^(NH)-Val-Cit-^(C═O) or ^(NH)-Val-Ala-^(C═O).

Other dipeptide combinations of interest include:

-   -   ^(NH)-Gly-Gly-^(C═O),     -   ^(NH)-Pro-Pro-^(C═O), and     -   ^(NH)-Val-Glu-^(C═O).

Other dipeptide combinations may be used, including those described by Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869, which is incorporated herein by reference.

In some embodiments, Q is a tripeptide residue. The amino acids in the tripeptide may be any combination of natural amino acids and non-natural amino acids. In some embodiments, the tripeptide comprises natural amino acids. Where the linker is a cathepsin labile linker, the tripeptide is the site of action for cathepsin-mediated cleavage. The tripeptide then is a recognition site for cathepsin.

In one embodiment, the amino acid side chain is chemically protected, where appropriate. The side chain protecting group may be a group as discussed above. Protected amino acid sequences are cleavable by enzymes. For example, a dipeptide sequence comprising a Boc side chain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known in the art and are described in the Novabiochem Catalog, and as described above.

In some embodiments, the groups Q and —C(═O)—X₁—NHC(═O)X₂—NH— may be the same.

In some embodiments, the groups Q and —C(═O)—X₁—NHC(═O)X₂—NH— may be different.

L and G

L is a linker connected to the cell binding agent in the conjugate compound. G is a linker for connecting the PBD dimer to the cell binding agent to form the conjugate compound.

Preferably, the linker contains an electrophilic functional group for reaction with a nucleophilic functional group on the cell binding agent. Nucleophilic groups on antibodies include, but are not limited to: (i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) maleimide groups (ii) activated disulfides, (iii) active esters such as NHS (N-hydroxysuccinimide) esters, HOBt (N-hydroxybenzotriazole) esters, haloformates, and acid halides; (iv) alkyl and benzyl halides such as haloacetamides; and (v) aldehydes, ketones, carboxyl, and, some of which are exemplified as follows:

Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541 teaches engineering antibodies by introduction of reactive cysteine amino acids. In some embodiments, a Linker has a reactive nucleophilic group which is reactive with an electrophilic group present on an antibody. Useful electrophilic groups on an antibody include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatom of a nucleophilic group of a Linker can react with an electrophilic group on an antibody and form a covalent bond to an antibody unit. Useful nucleophilic groups on a Linker include, but are not limited to, hydrazide, oxime, amino, hydroxyl, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. The electrophilic group on an antibody provides a convenient site for attachment to a Linker.

In one embodiment, the group L is:

-   -   where the asterisk indicates the point of attachment to the rest         of group Y, the wavy line indicates the point of attachment to         the cell binding agent, and m is 0 to 6. In one embodiment, m is         5.

In one embodiment, the connection between the cell binding agent and L is through a thiol residue of the cell binding agent and a maleimide group of L.

In one embodiment, the connection between the cell binding agent and L is:

-   -   where the asterisk indicates the point of attachment to the         remaining portion of the L group or the remaining portion of the         Y group and the wavy line indicates the point of attachment to         the remaining portion of the cell binding agent. In this         embodiment, the S atom is typically derived from the cell         binding agent.

In each of the embodiments above, an alternative functionality may be used in place of the maleimide-derived group shown below:

-   -   where the wavy line indicates the point of attachment to the         cell binding agent as before, and the asterisk indicates the         bond to the remaining portion of the L group or the remaining         portion of the Y group.

In one embodiment, the maleimide-derived group is replaced with the group:

-   -   where the wavy line indicates point of attachment to the cell         binding agent, and the asterisk indicates the bond to the         remaining portion of the L group or the remaining portion of the         Y group.

In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the cell binding agent, is selected from:

-   -   —C(═O)NH—,     -   —C(═O)O—,     -   —NHC(═O)—,     -   —OC(═O)—,     -   —OC(═O)O—,     -   —NHC(═O)O—,     -   —OC(═O)NH—,     -   —NHC(═O)NH—,     -   —NHC(═O)NH,     -   —C(═O)NHC(═O)—,     -   —S—,     -   —S—S—,     -   —CH₂C(═O)—     -   —C(═O)CH₂—,     -   ═N—NH—, and     -   —NH—N═.

In one embodiment, the maleimide-derived group is replaced with a group, which optionally together with the cell binding agent, is selected from:

-   -   where the wavy line indicates either the point of attachment to         the cell binding agent or the bond to the remaining portion of         the L group or the remaining portion of the Y group, and the         asterisk indicates the other of the point of attachment to the         cell binding agent or the bond to the remaining portion of the L         group or the remaining portion of the Y group.

Other groups that can be used as L for connecting the remaining portion of the Y group to the cell binding agent are described in WO 2005/082023.

Thus, in embodiments of the present invention, L is of formula: -L^(A)-(CH₂)_(m)—

Where m is from 0 to 6; and

L^(A) is selected from:

where Ar represents a C₅₋₆ arylene group, e.g. phenylene.

Thus, in embodiments of the present invention, G is of formula: G^(A)-(CH₂)_(m)—

Where m is from 0 to 6; and

G^(A) is selected from:

where Ar represents a C₅₋₆ arylene group, e.g. phenylene.

In some embodiments, m may be 2 or 5.

Cell Binding Agent

A cell binding agent may be of any kind, and include peptides and non-peptides. These can include antibodies or a fragment of an antibody that contains at least one binding site, lymphokines, hormones, hormone mimetics, vitamins, growth factors, nutrient-transport molecules, or any other cell binding molecule or substance.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptide comprising 4-30, preferably 6-20, contiguous amino acid residues. In this embodiment, it is preferred that one cell binding agent is linked to one monomer or dimer pyrrolobenzodiazepine compound.

In one embodiment the cell binding agent comprises a peptide that binds integrin α_(v)β₆. The peptide may be selective for α_(v)β₆ over XYS.

In one embodiment the cell binding agent comprises the A20FMDV-Cys polypeptide. The A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC. Alternatively, a variant of the A20FMDV-Cys sequence may be used wherein one, two, three, four, five, six, seven, eight, nine or ten amino acid residues are substituted with another amino acid residue. Furthermore, the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC.

Antibodies

The term “antibody” herein is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity (Miller et al (2003) Jour. of Immunology 170:4854-4861). Antibodies may be murine, human, humanized, chimeric, or derived from other species. An antibody is a protein generated by the immune system that is capable of recognizing and binding to a specific antigen. (Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York). A target antigen generally has numerous binding sites, also called epitopes, recognized by CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, one antigen may have more than one corresponding antibody. An antibody includes a full-length immunoglobulin molecule or an immunologically active portion of a full-length immunoglobulin molecule, i.e., a molecule that contains an antigen binding site that immunospecifically binds an antigen of a target of interest or part thereof, such targets including but not limited to, cancer cell or cells that produce autoimmune antibodies associated with an autoimmune disease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD, and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule. The immunoglobulins can be derived from any species, including human, murine, or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and scFv fragments; diabodies; linear antibodies; fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, CDR (complementary determining region), and epitope-binding fragments of any of the above which immunospecifically bind to cancer cell antigens, viral antigens or microbial antigens, single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e. the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al (1975) Nature 256:495, or may be made by recombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al (1991) Nature, 352:624-628; Marks et al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carrying a fully human immunoglobulin system (Lonberg (2008) Curr. Opinion 20(4):450-459).

The monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies include “primatized” antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g. Old World Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising a VL and VH domains, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more “effector functions” which refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down regulation of cell surface receptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different “classes.” There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into “subclasses” (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

Humanisation

Techniques to reduce the in vivo immunogenicity of a non-human antibody or antibody fragment include those termed “humanisation”.

A “humanized antibody” refers to a polypeptide comprising at least a portion of a modified variable region of a human antibody wherein a portion of the variable region, preferably a portion substantially less than the intact human variable domain, has been substituted by the corresponding sequence from a non-human species and wherein the modified variable region is linked to at least another part of another protein, preferably the constant region of a human antibody. The expression “humanized antibodies” includes human antibodies in which one or more complementarity determining region (“CDR”) amino acid residues and/or one or more framework region (“FW” or “FR”) amino acid residues are substituted by amino acid residues from analogous sites in rodent or other non-human antibodies. The expression “humanized antibody” also includes an immunoglobulin amino acid sequence variant or fragment thereof that comprises an FR having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Or, looked at another way, a humanized antibody is a human antibody that also contains selected sequences from non-human (e.g. murine) antibodies in place of the human sequences. A humanized antibody can include conservative amino acid substitutions or non-natural residues from the same or different species that do not significantly alter its binding and/or biologic activity.

Such antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins.

There are a range of humanisation techniques, including ‘CDR grafting’, ‘guided selection’, ‘deimmunization’, ‘resurfacing’ (also known as ‘veneering’), ‘composite antibodies’, ‘Human String Content Optimisation’ and framework shuffling.

CDR Grafting

In this technique, the humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary-determining region (CDR) of the recipient antibody are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, camel, bovine, goat, or rabbit having the desired properties (in effect, the non-human CDRs are ‘grafted’ onto the human framework). In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues (this may happen when, for example, a particular FR residue has significant effect on antigen binding).

Furthermore, humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and maximize antibody performance. Thus, in general, a humanized antibody will comprise all of at least one, and in one aspect two, variable domains, in which all or all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), or that of a human immunoglobulin.

Guided Selection

The method consists of combining the V_(H) or V_(L) domain of a given non-human antibody specific for a particular epitope with a human V_(H) or V_(L) library and specific human V domains are selected against the antigen of interest. This selected human VH is then combined with a VL library to generate a completely human VH×VL combination. The method is described in Nature Biotechnology (N.Y.) 12, (1994) 899-903.

Composite Antibodies

In this method, two or more segments of amino acid sequence from a human antibody are combined within the final antibody molecule. They are constructed by combining multiple human VH and VL sequence segments in combinations which limit or avoid human T cell epitopes in the final composite antibody V regions. Where required, T cell epitopes are limited or avoided by, exchanging V region segments contributing to or encoding a T cell epitope with alternative segments which avoid T cell epitopes. This method is described in US 2008/0206239 A1.

Deimmunization

This method involves the removal of human (or other second species) T-cell epitopes from the V regions of the therapeutic antibody (or other molecule). The therapeutic antibodies V-region sequence is analysed for the presence of MHC class II-binding motifs by, for example, comparison with databases of MHC-binding motifs (such as the “motifs” database hosted at www.wehi.edu.au). Alternatively, MHC class II-binding motifs may be identified using computational threading methods such as those devised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in these methods, consecutive overlapping peptides from the V-region sequences are testing for their binding energies to MHC class II proteins. This data can then be combined with information on other sequence features which relate to successfully presented peptides, such as amphipathicity, Rothbard motifs, and cleavage sites for cathepsin B and other processing enzymes.

Once potential second species (e.g. human) T-cell epitopes have been identified, they are eliminated by the alteration of one or more amino acids. The modified amino acids are usually within the T-cell epitope itself, but may also be adjacent to the epitope in terms of the primary or secondary structure of the protein (and therefore, may not be adjacent in the primary structure). Most typically, the alteration is by way of substitution but, in some circumstances amino acid addition or deletion will be more appropriate.

All alterations can be accomplished by recombinant DNA technology, so that the final molecule may be prepared by expression from a recombinant host using well established methods such as Site Directed Mutagenesis. However, the use of protein chemistry or any other means of molecular alteration is also possible.

Resurfacing

This method involves:

-   -   (a) determining the conformational structure of the variable         region of the non-human (e.g. rodent) antibody (or fragment         thereof) by constructing a three-dimensional model of the         non-human antibody variable region;     -   (b) generating sequence alignments using relative accessibility         distributions from x-ray crystallographic structures of a         sufficient number of non-human and human antibody variable         region heavy and light chains to give a set of heavy and light         chain framework positions wherein the alignment positions are         identical in 98% of the sufficient number of non-human antibody         heavy and light chains;     -   (c) defining for the non-human antibody to be humanized, a set         of heavy and light chain surface exposed amino acid residues         using the set of framework positions generated in step (b);     -   (d) identifying from human antibody amino acid sequences a set         of heavy and light chain surface exposed amino acid residues         that is most closely identical to the set of surface exposed         amino acid residues defined in step (c), wherein the heavy and         light chain from the human antibody are or are not naturally         paired;     -   (e) substituting, in the amino acid sequence of the non-human         antibody to be humanized, the set of heavy and light chain         surface exposed amino acid residues defined in step (c) with the         set of heavy and light chain surface exposed amino acid residues         identified in step (d);     -   (f) constructing a three-dimensional model of the variable         region of the non-human antibody resulting from the substituting         specified in step (e);     -   (g) identifying, by comparing the three-dimensional models         constructed in steps (a) and (f), any amino acid residues from         the sets identified in steps (c) or (d), that are within 5         Angstroms of any atom of any residue of the complementarity         determining regions of the non-human antibody to be humanized;         and     -   (h) changing any residues identified in step (g) from the human         to the original non-human amino acid residue to thereby define a         non-human antibody humanizing set of surface exposed amino acid         residues; with the proviso that step (a) need not be conducted         first, but must be conducted prior to step (g).         Superhumanization

The method compares the non-human sequence with the functional human germline gene repertoire. Those human genes encoding canonical structures identical or closely related to the non-human sequences are selected. Those selected human genes with highest homology within the CDRs are chosen as FR donors. Finally, the non-human CDRs are grafted onto these human FRs. This method is described in patent WO 2005/079479 A2.

Human String Content Optimization

This method compares the non-human (e.g. mouse) sequence with the repertoire of human germline genes and the differences are scored as Human String Content (HSC) that quantifies a sequence at the level of potential MHC/T-cell epitopes. The target sequence is then humanized by maximizing its HSC rather than using a global identity measure to generate multiple diverse humanized variants (described in Molecular Immunology, 44, (2007) 1986-1998).

Framework Shuffling

The CDRs of the non-human antibody are fused in-frame to cDNA pools encompassing all known heavy and light chain human germline gene frameworks. Humanised antibodies are then selected by e.g. panning of the phage displayed antibody library. This is described in Methods 36, 43-60 (2005).

Examples of cell binding agents include those agents described for use in WO 2007/085930, which is incorporated herein.

Tumour-associate antigens and cognate antibodies for use in embodiments of the present invention are listed below.

Tumor-Associated Antigens and Cognate Antibodies

(1) BMPR1B (Bone Morphogenetic Protein Receptor-Type IB)

Nucleotide

-   Genbank accession no. NM_001203 -   Genbank version no. NM_001203.2 GI:169790809 -   Genbank record update date: Sep. 23, 2012 02:06 PM     Polypeptide -   Genbank accession no. NP 001194 -   Genbank version no. NP 001194.1 GI:4502431 -   Genbank record update date: Sep. 23, 2012 02:06 PM     Cross-References -   ten Dijke, P., et al Science 264 (5155): 101-104 (1994), Oncogene 14     10 (11):1377-1382 (1997)); WO2004/063362 (Claim 2); WO2003/042661     (Claim 12); US2003/134790-A1 (Page 38-39); WO2002/102235 (Claim 13;     Page 296); WO2003/055443 (Page 91-92); WO2002/99122 (Example 2; Page     528-530); WO2003/029421 (Claim 6); WO2003/024392 (Claim 2; FIG.     112); WO2002/98358 (Claim 1; Page 183); WO2002/54940 (Page 100-101);     WO2002/59377 (Page 349-350); WO2002/30268 (Claim 27; Page 376); 15     WO2001/48204 (Example; FIG. 4); NP_001194 bone morphogenetic protein     receptor, type IB/pid=NP_001194.1; MIM:603248; AY065994     (2) E16 (LAT1, SLC7A5)     Nucleotide -   Genbank accession no. NM_003486 -   Genbank version no. NM_003486.5 GI:71979931 -   Genbank record update date: Jun. 27, 2012 12:06 PM     Polypeptide -   Genbank accession no. NP_003477 -   Genbank version no. NP_003477.4 GI:71979932 -   Genbank record update date: Jun. 27, 2012 12:06 PM     Cross References -   Biochem. Biophys. Res. -   Commun. 255 (2), 283-288 (1999), Nature 395 (6699):288-291 (1998),     Gaugitsch, H. W., et 20 al (1992) J. Biol. Chem. 267     (16):11267-11273); WO2004/048938 (Example 2); WO2004/032842 (Example     IV); WO2003/042661 (Claim 12); WO2003/016475 (Claim 1); WO2002/78524     (Example 2); WO2002/99074 (Claim 19; Page 127-129); WO2002/86443     (Claim 27; Pages 222, 393); WO2003/003906 (Claim 10; Page 293);     WO2002/64798 (Claim 33; Page 93-95); WO2000/14228 (Claim 5; Page     133-136); US2003/224454 (FIG. 3); 25 WO2003/025138 (Claim 12; Page     150); NP_003477 solute carrier family 7 (cationic amino -   acid transporter, y+system), member 5/pid=NP_003477.3—Homo sapiens;     MIM:600182; NM_015923.     (3) STEAP1 (Six Transmembrane Epithelial Antigen of Prostate)     Nucleotide -   Genbank accession no. NM_012449 -   Genbank version no. NM_012449.2 GI:22027487 -   Genbank record update date: Sep. 9, 2012 02:57 PM     Polypeptide -   Genbank accession no. NP_036581 -   Genbank version no. NP_036581.1 GI:9558759 -   Genbank record update date: Sep. 9, 2012 02:57 PM     Cross References -   Cancer Res. 61 (15), 5857-5860 (2001), Hubert, R. S., et al (1999)     Proc. Natl. Acad. Sci. U.S.A. 96 (25):14523-14528); WO2004/065577     (Claim 6); WO2004/027049 (FIG. 1L); EP1394274 (Example 11);     WO2004/016225 (Claim 2); WO2003/042661 (Claim 12); US2003/157089     (Example 5); US2003/185830 (Example 5); US2003/064397 (FIG. 2);     WO2002/89747 (Example 5; Page 618-619); WO2003/022995 (Example 9;     FIG. 13A, Example 53; Page 173, Example 2; FIG. 2A); six     transmembrane epithelial antigen of the prostate; MIM:604415.     (4) 0772P (CA125, MUC16)     Nucleotide -   Genbank accession no. AF361486 -   Genbank version no. AF361486.3 GI:34501466 -   Genbank record update date: Mar. 11, 2010 07:56 AM     Polypeptide -   Genbank accession no. AAK74120 -   Genbank version no. AAK74120.3 GI:34501467 -   Genbank record update date: Mar. 11, 2010 07:56 AM     Cross References -   J. Biol. Chem. 276 (29):27371-27375 (2001)); WO2004/045553 (Claim     14); WO2002/92836 (Claim 6; FIG. 12); WO2002/83866 (Claim 15; Page     116-121); US2003/124140 (Example 16); GI:34501467;     (5) MPF (MPF, MSLN, SMR, Megakaryocyte Potentiating Factor,     Mesothelin)     Nucleotide -   Genbank accession no. NM_005823 -   Genbank version no. NM_005823.5 GI:293651528 -   Genbank record update date: Sep. 2, 2012 01:47 PM     Polypeptide -   Genbank accession no. NP_005814 -   Genbank version no. NP_005814.2 GI:53988378 -   Genbank record update date: Sep. 2, 2012 01:47 PM     Cross References -   Yamaguchi, N., et al Biol. Chem. 269 (2), 805-808 (1994), Proc.     Natl. Acad. Sci. U.S.A. 96 (20):11531-11536 (1999), Proc. Natl.     Acad. Sci. U.S.A. 93 10 (1):136-140 (1996), J. Biol. Chem. 270     (37):21984-21990 (1995)); WO2003/101283 (Claim 14); (WO2002/102235     (Claim 13; Page 287-288); WO2002/101075 (Claim 4; Page 308-309);     WO2002/71928 (Page 320-321); WO94/10312 (Page 52-57); IM:601051.     (6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, Solute Carrier Family 34     (Sodium Phosphate), Member 2, Type II Sodium-Dependent Phosphate     Transporter 3b)     Nucleotide -   Genbank accession no. NM_006424 -   Genbank version no. NM_006424.2 GI:110611905 -   Genbank record update date: Jul. 22, 2012 03:39 PM     Polypeptide -   Genbank accession no. NP_006415 -   Genbank version no. NP_006415.2 GI:110611906 -   Genbank record update date: Jul. 22, 2012 03:39 PM     Cross References -   J. Biol. Chem. 277 (22):19665-19672 (2002), Genomics 62 (2):281-284     (1999), Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun.     258 (3):578-582); WO2004/022778 (Claim 2); EP1394274 (Example 11);     WO2002/102235 (Claim 13; Page 20 326); EP0875569 (Claim 1; Page     17-19); WO2001/57188 (Claim 20; Page 329); WO2004/032842 (Example     IV); WO2001/75177 (Claim 24; Page 139-140); MIM:604217.     (7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin     5b Hlog, 25 Sema Domain, Seven Thrombospondin Repeats (Type 1 and     Type 1-Like), Transmembrane Domain™ and Short Cytoplasmic Domain,     (Semaphorin) 5B)     Nucleotide -   Genbank accession no. AB040878 -   Genbank version no. AB040878.1 GI:7959148 -   Genbank record update date: Aug. 2, 2006 05:40 PM     Polypeptide -   Genbank accession no. BAA95969 -   Genbank version no. BAA95969.1 GI:7959149 -   Genbank record update date: Aug. 2, 2006 05:40 PM     Cross References -   Nagase T., et al (2000) DNA Res. 7 (2):143-150); WO2004/000997     (Claim 1); WO2003/003984 (Claim 1); WO2002/06339 (Claim 1; Page 50);     WO2001/88133 (Claim 1; Page 41-43, 48-58); WO2003/054152 (Claim 20);     WO2003/101400 (Claim 11); Accession: 30 Q9P283; Genew; HGNC:10737     (8) PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12,     RIKEN cDNA 2700050C12 Gene)     Nucleotide -   Genbank accession no. AY358628 -   Genbank version no. AY358628.1 GI:37182377 -   Genbank record update date: Dec. 1, 2009 04:15 AM     Polypeptide -   Genbank accession no. AAQ88991 -   Genbank version no. AAQ88991.1 GI:37182378 -   Genbank record update date: Dec. 1, 2009 04:15 AM     Cross References -   Ross et al (2002) Cancer Res. 62:2546-2553; US2003/129192 (Claim 2);     US2004/044180 (Claim 12); US2004/044179 35 (Claim 11); US2003/096961     (Claim 11); US2003/232056 (Example 5); WO2003/105758 16 (Claim 12);     US2003/206918 (Example 5); EP1347046 (Claim 1); WO2003/025148 (Claim     20); GI:37182378.     (9) ETBR (Endothelin Type B Receptor)     Nucleotide -   Genbank accession no. AY275463 -   Genbank version no. AY275463.1 GI:30526094 -   Genbank record update date: Mar. 11, 2010 02:26 AM     Polypeptide -   Genbank accession no. AAP32295 -   Genbank version no. AAP32295.1 GI:30526095 -   Genbank record update date: Mar. 11, 2010 02:26 AM     Cross References -   Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991;     Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991;     Arai H., et al Jpn. Circ. J. 56, 1303-1307, 1992; Arai H., et al J.     Biol. Chem. 268, 3463-3470, 1993; Sakamoto A., Yanagisawa M., et al     Biochem. Biophys. Res. Commun. 178, 656-663, 1991; Elshourbagy N.     A., et al J. Biol. Chem. 268, 3873-3879, 1993; Haendler B., et al J.     Cardiovasc. Pharmacol. 20, s1-S4, 1992; Tsutsumi M., et al Gene 228,     43-49, 1999; Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A.     99, 16899-16903, 2002; Bourgeois C., et al J. Clin. Endocrinol.     Metab. 82, 3116-3123, 1997; -   Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997; Verheij J. B.,     et al Am. J. Med. Genet. 108, 223-225, 2002; Hofstra R. M. W., et al     Eur. J. Hum. Genet. 5, 180-185, 1997; Puffenberger E. G., et al Cell     79, 1257-1266, 1994; Attie T., et al, Hum. Mol. Genet. 4, 2407-15     2409, 1995; Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996;     Amiel J., et al Hum. Mol. -   Genet. 5, 355-357, 1996; Hofstra R. M. W., et al Nat. Genet. 12,     445-447, 1996; Svensson P. J., et al Hum. Genet. 103, 145-148, 1998;     Fuchs S., et al Mol. Med. 7, 115-124, 2001; Pingault V., et     al (2002) Hum. Genet. 111, 198-206; WO2004/045516 (Claim 1);     WO2004/048938 (Example 2); WO2004/040000 (Claim 151); WO2003/087768     (Claim 1); 20 WO2003/016475 (Claim 1); WO2003/016475 (Claim 1);     WO2002/61087 (FIG. 1); WO2003/016494 (FIG. 6); WO2003/025138 (Claim     12; Page 144); WO2001/98351 (Claim 1; Page 124-125); EP0522868     (Claim 8; FIG. 2); WO2001/77172 (Claim 1; Page 297-299);     US2003/109676; U.S. Pat. No. 6,518,404 (FIG. 3); U.S. Pat. No.     5,773,223 (Claim 1a; Col 31-34); WO2004/001004.     (10) MSG783 (RNF124, Hypothetical Protein FLJ20315)     Nucleotide -   Genbank accession no. NM_017763 -   Genbank version no. NM_017763.4 GI:167830482 -   Genbank record update date: Jul. 22, 2012 12:34 AM     Polypeptide -   Genbank accession no. NP_060233 -   Genbank version no. NP_060233.3 GI:56711322 -   Genbank record update date: Jul. 22, 2012 12:34 AM     Cross References -   WO2003/104275 (Claim 1); WO2004/046342 (Example 2); WO2003/042661     (Claim 12); WO2003/083074 (Claim 14; Page 61); WO2003/018621 (Claim     1); WO2003/024392 (Claim 2; FIG. 93); WO2001/66689 (Example 6);     LocusID:54894.     (11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP,     Prostate Cancer Associated Gene 1, Prostate Cancer Associated     Protein 1, Six Transmembrane Epithelial Antigen of Prostate 2, Six     Transmembrane Prostate Protein)     Nucleotide -   Genbank accession no. AF455138 -   Genbank version no. AF455138.1 GI:22655487 -   Genbank record update date: Mar. 11, 2010 01:54 AM     Polypeptide -   Genbank accession no. AAN04080 -   Genbank version no. AAN04080.1 GI:22655488 -   Genbank record update date: Mar. 11, 2010 01:54 AM     Cross References -   Lab. Invest. 82 (11):1573-1582 (2002)); WO2003/087306; US2003/064397     (Claim 1; FIG. 1); WO2002/72596 (Claim 13; Page 54-55); WO2001/72962     (Claim 1; FIG. 4B); 35 WO2003/104270 (Claim 11); WO2003/104270     (Claim 16); US2004/005598 (Claim 22); WO2003/042661 (Claim 12);     US2003/060612 (Claim 12; FIG. 10); WO2002/26822 (Claim 23; FIG. 2);     WO2002/16429 (Claim 12; FIG. 10); GI:22655488.     (12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, Transient Receptor     Potential Cation 5 Channel, Subfamily M, Member 4)     Nucleotide -   Genbank accession no. NM_017636 -   Genbank version no. NM_017636.3 GI:304766649 -   Genbank record update date: Jun. 29, 2012 11:27 AM     Polypeptide -   Genbank accession no. NP_060106 -   Genbank version no. NP_060106.2 GI:21314671 -   Genbank record update date: Jun. 29, 2012 11:27 AM     Cross References -   Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697     (2001), Cell 109 (3):397-407 (2002), J. Biol. Chem. 278     (33):30813-30820 (2003)); US2003/143557 (Claim 4); WO2000/40614     (Claim 14; Page 100-103); WO2002/10382 (Claim 1; FIG. 9A);     WO2003/042661 (Claim 12); WO2002/30268 (Claim 27; Page 391);     US2003/219806 (Claim 4); WO2001/62794 (Claim 10 14; FIG. 1A-D);     MIM:606936.     (13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, Teratocarcinoma-Derived     Growth Factor)     Nucleotide -   Genbank accession no. NM_003212 -   Genbank version no. NM_003212.3 GI:292494881 -   Genbank record update date: Sep. 23, 2012 02:27 PM     Polypeptide -   Genbank accession no. NP_003203 -   Genbank version no. NP_003203.1 GI:4507425 -   Genbank record update date: Sep. 23, 2012 02:27 PM     Cross References -   Ciccodicola, A., et al EMBO J. 8 (7):1987-1991 (1989), Am. J. Hum.     Genet. 49 (3):555-565 (1991)); US2003/224411 (Claim 1);     WO2003/083041 (Example 1); WO2003/034984 (Claim 12); WO2002/88170     (Claim 2; Page 52-53); WO2003/024392 (Claim 2; FIG. 58);     WO2002/16413 (Claim 1; Page 94-95, 105); WO2002/22808 (Claim 2; FIG.     1); U.S. Pat. No. 5,854,399 (Example 2; Col 17-18); U.S. Pat. No.     5,792,616 (FIG. 2); MIM:187395.     (14) CD21 (CR2 (Complement Receptor 2) or C3DR (C3d/Epstein Barr     Virus Receptor) or Hs.73792)     Nucleotide -   Genbank accession no M26004 -   Genbank version no. M26004.1 GI:181939 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Polypeptide -   Genbank accession no. AAA35786 -   Genbank version no. AAA35786.1 GI:181940 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Cross References -   Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125); Weis J. J.,     et al J. Exp. Med. 167, 1047-1066, 1988; Moore M., et al Proc. Natl.     Acad. Sci. U.S.A. 84, 9194-9198, 1987; Barel M., et al Mol. Immunol.     35, 1025-1031, 1998; Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A.     83, 5639-5643, 1986; Sinha S. K., et al (1993) J. Immunol. 150,     5311-5320; WO2004/045520 (Example 4); US2004/005538 (Example 1);     WO2003/062401 (Claim 9); WO2004/045520 (Example 4); WO91/02536     (FIGS. 9.1-9.9); WO2004/020595 (Claim 1); Accession: P20023; Q13866;     Q14212; EMBL; M26004; AAA35786.1.     (15) CD79b (CD79B, CD79β, IGb (Immunoglobulin-Associated Beta), B29)     Nucleotide -   Genbank accession no NM_000626 -   Genbank version no. NM_000626.2 GI:90193589 -   Genbank record update date: Jun. 26, 2012 01:53 PM     Polypeptide -   Genbank accession no. NP_000617 -   Genbank version no. NP_000617.1 GI:11038674 -   Genbank record update date: Jun. 26, 2012 01:53 PM     Cross References -   Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131, Blood (2002)     100 (9):3068-3076, Muller et al (1992) Eur. J. Immunol. 22     (6):1621-1625); WO2004/016225 (claim 2, FIG. 140); WO2003/087768,     US2004/101874 (claim 1, page 102); WO2003/062401 (claim 9);     WO2002/78524 (Example 2); US2002/150573 (claim 35 5, page 15); U.S.     Pat. No. 5,644,033; WO2003/048202 (claim 1, pages 306 and 309); WO     99/58658, U.S. Pat. No. 6,534,482 (claim 13, FIG. 17A/B);     WO2000/55351 (claim 11, pages 1145-1146); MIM:147245     (16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 Domain Containing Phosphatase     Anchor Protein 5 1a), SPAP1B, SPAP1C)     Nucleotide -   Genbank accession no NM_030764 -   Genbank version no. NM_030764.3 GI:227430280 -   Genbank record update date: Jun. 30, 2012 12:30 AM     Polypeptide -   Genbank accession no. NP_110391 -   Genbank version no. NP_110391.2 GI:19923629 -   Genbank record update date: Jun. 30, 2012 12:30 AM     Cross References -   AY358130); Genome Res. 13 (10):2265-2270 (2003), Immunogenetics 54     (2):87-95 (2002), Blood 99 (8):2662-2669 (2002), Proc. Natl. Acad.     Sci. U.S.A. 98 (17):9772-9777 (2001), Xu, M. J., et al (2001)     Biochem. Biophys. Res. Commun. 280 (3):768-775; WO2004/016225 (Claim     2); WO2003/077836; WO2001/38490 (Claim 5; FIG. 18D-1-18D-2);     WO2003/097803 (Claim 12); -   10 WO2003/089624 (Claim 25); MIM:606509.     (17) HER2 (ErbB2)     Nucleotide -   Genbank accession no M11730 -   Genbank version no. M11730.1 GI:183986 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Polypeptide -   Genbank accession no. AAA75493 -   Genbank version no. AAA75493.1 GI:306840 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Cross References -   Coussens L., et al Science (1985) 230(4730):1132-1139); Yamamoto T.,     et al Nature 319, 230-234, 1986; Semba K., et al Proc. Natl. Acad.     Sci. U.S.A. 82, 6497-6501, 1985; Swiercz J. M., et al J. Cell Biol.     165, 869-15 880, 2004; Kuhns J. J., et al J. Biol. Chem. 274,     36422-36427, 1999; Cho H.-S., et al Nature 421, 756-760, 2003;     Ehsani A., et al (1993) Genomics 15, 426-429; WO2004/048938 (Example     2); WO2004/027049 (FIG. 11); WO2004/009622; WO2003/081210; -   WO2003/089904 (Claim 9); WO2003/016475 (Claim 1); US2003/118592;     WO2003/008537 (Claim 1); WO2003/055439 (Claim 29; FIG. 1A-B);     WO2003/025228 (Claim 37; FIG. 5C); 20 WO2002/22636 (Example 13; Page     95-107); WO2002/12341 (Claim 68; FIG. 7); WO2002/13847 (Page 71-74);     WO2002/14503 (Page 114-117); WO2001/53463 (Claim 2; Page 41-46);     WO2001/41787 (Page 15); WO2000/44899 (Claim 52; FIG. 7);     WO2000/20579 (Claim 3; FIG. 2); U.S. Pat. No. 5,869,445 (Claim 3;     Col 31-38); WO9630514 (Claim 2; Page 56-61); EP1439393 (Claim 7);     WO2004/043361 (Claim 7); WO2004/022709; WO2001/00244 25 (Example 3;     FIG. 4); Accession: P04626; EMBL; M11767; AAA35808.1. EMBL; M11761;     AAA35808.1     Antibodies -   Abbott: US20110177095     -   For example, an antibody comprising CDRs having overall at least         80% sequence identity to CDRs having amino acid sequences of SEQ         ID NO:3 (CDR-H1), SEQ ID NO:4 (CDR-H2), SEQ ID NO:5 (CDR-H3),         SEQ ID NO:104 and/or SEQ ID NO:6 (CDR-L1), SEQ ID NO:7 (CDR-L2),         and SEQ ID NO:8 (CDR-L3), wherein the anti-HER2 antibody or         anti-HER2 binding fragment has reduced immunogenicity as         compared to an antibody having a VH of SEQ ID NO:1 and a VL of         SEQ ID NO:2. -   Biogen: US20100119511     -   For example, ATCC accession numbers: PTA-10355, PTA-10356,         PTA-10357, PTA-10358     -   For example, a purified antibody molecule that binds to HER2         comprising a all six CDR's from an antibody selected from the         group consisting of BIIB71F10 (SEQ ID NOs:11, 13), BIIB69A09         (SEQ ID NOs:15, 17); BIIB67F10 (SEQ ID NOs:19, 21); BIIB67F11         (SEQ ID NOs:23, 25), BIIB66A12 (SEQ ID NOs:27, 29), BIIB66C01         (SEQ ID NOs:31, 33), BIIB65C10 (SEQ ID NOs:35, 37), BIIB65H09         (SEQ ID NOs:39, 41) and BIIB65B03 (SEQ ID NOs:43, 45), or CDRs         which are identical or which have no more than two alterations         from said CDRs. -   Herceptin (Genentech)—U.S. Pat. No. 6,054,297; ATCC accession no.     CRL-10463 (Genentech) -   Pertuzumab (Genentech)     -   US20110117097         -   for example, see SEQ IDs No. 15&16, SEQ IDs No. 17&18, SEQ             IDs No. 23&24 & ATCC accession numbers HB-12215, HB-12216,             CRL 10463, HB-12697.     -   US20090285837     -   US20090202546         -   for example, ATCC accession numbers: HB-12215, HB-12216, CRL             10463, HB-12698.     -   US20060088523         -   for example, ATCC accession numbers: HB-12215, HB-12216         -   for example, an antibody comprising the variable light and             variable heavy amino acid sequences in SEQ ID Nos. 3 and 4,             respectively.         -   for example, an antibody comprising a light chain amino acid             sequence selected from SEQ ID No. 15 and 23, and a heavy             chain amino acid sequence selected from SEQ ID No. 16 and 24     -   US20060018899         -   for example, ATCC accession numbers: (7C2) HB-12215, (7F3)             HB-12216, (4D5) CRL-10463, (2C4) HB-12697.         -   for example, an antibody comprising the amino acid sequence             in SEQ ID No. 23, or a deamidated and/or oxidized variant             thereof.     -   US2011/0159014         -   for example, an antibody having a light chain variable             domain comprising the hypervariable regions of SEQ ID NO:             1″.         -   For example, an antibody having a heavy chain variable             domain comprising the hypervariable regions of SEQ ID NO: 2.     -   US20090187007 -   Glycotope: TrasGEX antibody http://www.glycotope.com/pipeline     -   For example, see International Joint Cancer Institute and         Changhai Hospital Cancer Cent: HMTI-Fc Ab—Gao J., et al BMB Rep.         2009 Oct. 31; 42(10):636-41. -   Symphogen: US20110217305 -   Union Stem Cell &Gene Engineering, China—Liu H Q., et al Xi Bao Yu     Fen Zi Mian YiXue Za Zhi. 2010 May; 26(5):456-8.     (18) NCA (CEACAM6)     Nucleotide -   Genbank accession no M18728 -   Genbank version no. M18728.1 GI:189084 -   Genbank record update date: Jun. 23, 2010 08:48 AM     Polypeptide -   Genbank accession no. AAA59907 -   Genbank version no. AAA59907.1 GI:189085 -   Genbank record update date: Jun. 23, 2010 08:48 AM     Cross References -   Barnett T., et al Genomics 3, 59-66, 1988; Tawaragi Y., et al     Biochem. Biophys. Res. Commun. 150, 89-96, 1988; Strausberg R. L.,     et al Proc. Natl. Acad. Sci. U.S.A. 99:16899-16903, 2002;     WO2004/063709; EP1439393 (Claim 7); WO2004/044178 (Example 4);     WO2004/031238; WO2003/042661 (Claim 12); WO2002/78524 (Example 2);     WO2002/86443 (Claim 27; Page 427); WO2002/60317 (Claim 2);     Accession: P40199; Q14920; EMBL; M29541; AAA59915.1. -   EMBL; M18728.     (19) MDP (DPEP1)     Nucleotide -   Genbank accession no BC017023 -   Genbank version no. BC017023.1 GI:16877538 -   Genbank record update date: Mar. 6, 2012 01:00 PM     Polypeptide -   Genbank accession no. AAH17023 -   Genbank version no. AAH17023.1 GI:16877539 -   Genbank record update date: Mar. 6, 2012 01:00 PM     Cross References -   Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002));     WO2003/016475 (Claim 1); WO2002/64798 (Claim 33; Page 85-87);     JP05003790 (FIG. 6-8); WO99/46284 (FIG. 9); MIM:179780.     (20) IL20R-alpha (IL20Ra, ZCYTOR7)     Nucleotide -   Genbank accession no AF184971 -   Genbank version no. AF184971.1 GI:6013324 -   Genbank record update date: Mar. 10, 2010 10:00 PM     Polypeptide -   Genbank accession no. AAF01320 -   Genbank version no. AAF01320.1 GI:6013325 -   Genbank record update date: Mar. 10, 2010 10:00 PM     Cross References -   Clark H. F., et al Genome Res. 13, 2265-2270, 2003; Mungall A. J.,     et al Nature 425, 805-811, 2003; Blumberg H., et al Cell 104, 9-19,     2001; Dumoutier L., et al J. Immunol. 167, 3545-3549, -   2001; Parrish-Novak J., et al J. Biol. Chem. 277, 47517-47523, 2002;     Pletnev S., et al (2003) 10 Biochemistry 42:12617-12624; Sheikh F.,     et al (2004) J. Immunol. 172, 2006-2010; EP1394274 (Example 11);     US2004/005320 (Example 5); WO2003/029262 (Page 74-75); WO2003/002717     (Claim 2; Page 63); WO2002/22153 (Page 45-47); US2002/042366 (Page     20-21); WO2001/46261 (Page 57-59); WO2001/46232 (Page 63-65);     WO98/37193 (Claim 1; Page 55-59); Accession: Q9UHF4; Q6UWA9; Q96SH8;     EMBL; AF184971; AAF01320.1.     (21) Brevican (BCAN, BEHAB)     Nucleotide -   Genbank accession no AF229053 -   Genbank version no. AF229053.1 GI:10798902 -   Genbank record update date: Mar. 11, 2010 12:58 AM     Polypeptide -   Genbank accession no. AAG23135 -   Genbank version no. AAG23135.1 GI:10798903 -   Genbank record update date: Mar. 11, 2010 12:58 AM     Cross References -   Gary S. C., et al Gene 256, 139-147, 2000; Clark H. F., et al Genome     Res. 13, 2265-2270, 2003; Strausberg R. L., et al Proc. Natl. Acad.     Sci. U.S.A. 99, 16899-16903, 2002; US2003/186372 (Claim 11);     US2003/186373 (Claim 11); US2003/119131 (Claim 1; FIG. 52);     US2003/119122 (Claim 1; 20 FIG. 52); US2003/119126 (Claim 1);     US2003/119121 (Claim 1; FIG. 52); US2003/119129 (Claim 1);     US2003/119130 (Claim 1); US2003/119128 (Claim 1; FIG. 52);     US2003/119125 (Claim 1); WO2003/016475 (Claim 1); WO2002/02634     (Claim 1)     (22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5)     Nucleotide -   Genbank accession no NM_004442 -   Genbank version no. NM_004442.6 GI:111118979 -   Genbank record update date: Sep. 8, 2012 04:43 PM     Polypeptide -   Genbank accession no. NP_004433 -   Genbank version no. NP_004433.2 GI:21396504 -   Genbank record update date: Sep. 8, 2012 04:43 PM     Cross References -   Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991) Oncogene     10 (5):897-905 (1995), Annu. Rev. Neurosci. 21:309-345 (1998), Int.     Rev. Cytol. 196:177-244 (2000)); WO2003042661 (Claim 12);     WO200053216 (Claim 1; Page 41); WO2004065576 (Claim 1); WO2004020583     (Claim 9); WO2003004529 (Page 128-132); WO200053216 (Claim 1; Page     42); MIM:600997.     (23) ASLG659 (B7h)     Nucleotide -   Genbank accession no. AX092328 -   Genbank version no. AX092328.1 GI:13444478 -   Genbank record update date: Jan. 26, 2011 07:37 AM     Cross References -   US2004/0101899 (Claim 2); WO2003104399 (Claim 11); WO2004000221     (FIG. 3); US2003/165504 (Claim 1); US2003/124140 (Example 2);     US2003/065143 (FIG. 60); WO2002/102235 (Claim 13; Page 299);     US2003/091580 (Example 2); WO2002/10187 (Claim 6; FIG. 10);     WO2001/94641 (Claim 12; FIG. 7b); WO2002/02624 (Claim 13; FIG.     1A-1B); US2002/034749 (Claim 54; Page 45-46); WO2002/06317 (Example     2; Page 320-321, Claim 34; Page 321-322); WO2002/71928 (Page     468-469); WO2002/02587 (Example 1; FIG. 1); WO2001/40269 (Example 3;     Pages 190-192); WO2000/36107 (Example 2; Page 205-207);     WO2004/053079 (Claim 12); WO2003/004989 (Claim 1); WO2002/71928     (Page 233-234, 452-453); WO 01/16318.     (24) PSCA (Prostate Stem Cell Antigen Precursor)     Nucleotide -   Genbank accession no AJ297436 -   Genbank version no. AJ297436.1 GI:9367211 -   Genbank record update date: Feb. 1, 2011 11:25 AM     Polypeptide -   Genbank accession no. CAB97347 -   Genbank version no. CAB97347.1 GI:9367212 -   Genbank record update date: Feb. 1, 2011 11:25 AM     Cross References -   Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740,     1998; Gu Z., et al Oncogene 19, -   1288-1296, 2000; Biochem. Biophys. Res. Commun. (2000)     275(3):783-788; WO2004/022709; EP1394274 (Example 11); US2004/018553     (Claim 17); WO2003/008537 (Claim 1); WO2002/81646 (Claim 1; Page     164); WO2003/003906 (Claim 10; Page 288); WO2001/40309 (Example 1;     FIG. 17); US2001/055751 (Example 1; FIG. 1b); WO2000/32752 (Claim     18; FIG. 1); WO98/51805 (Claim 17; Page 97); WO98/51824 (Claim 10;     Page 94); WO98/40403 (Claim 2; FIG. 1B); Accession: 043653; EMBL;     AF043498; AAC39607.1     (25) GEDA     Nucleotide -   Genbank accession no AY260763 -   Genbank version no. AY260763.1 GI:30102448 -   Genbank record update date: Mar. 11, 2010 02:24 AM     Polypeptide -   Genbank accession no. AAP14954 -   Genbank version no. AAP14954.1 GI:30102449 -   Genbank record update date: Mar. 11, 2010 02:24 AM     Cross References -   AP14954 lipoma HMGIC fusion-partnerlike protein/pid=AAP14954.1—Homo     sapiens (human); WO2003/054152 (Claim 20); WO2003/000842 (Claim 1);     WO2003/023013 (Example 3, Claim 20); US2003/194704 (Claim 45);     GI:30102449;     (26) BAFF-R (B Cell-Activating Factor Receptor, BLyS Receptor 3,     BR3)     Nucleotide -   Genbank accession no AF116456 -   Genbank version no. AF116456.1 GI:4585274 -   Genbank record update date: Mar. 10, 2010 09:44 PM     Polypeptide -   Genbank accession no. AAD25356 -   Genbank version no. AAD25356.1 GI:4585275 -   Genbank record update date: Mar. 10, 2010 09:44 PM     Cross References -   BAFF receptor/pid=NP_443177.1—Homo sapiens: Thompson, J. S., et al     Science 293 (5537), 2108-2111 (2001); WO2004/058309; WO2004/011611;     WO2003/045422 (Example; Page 32-33); WO2003/014294 (Claim 35; FIG.     6B); WO2003/035846 (Claim 70; Page 615-616); WO2002/94852 (Col     136-137); WO2002/38766 25 (Claim 3; Page 133); WO2002/24909 (Example     3; FIG. 3); MIM:606269; NP_443177.1; NM_052945_1; AF132600     (27) CD22 (B-Cell Receptor CD22-B Isoform, BL-CAM, Lyb-8, Lyb8,     SIGLEC-2, FLJ22814)     Nucleotide -   Genbank accession no AK026467 -   Genbank version no. AK026467.1 GI:10439337 -   Genbank record update date: Sep. 11, 2006 11:24 PM     Polypeptide -   Genbank accession no. BAB15489 -   Genbank version no. BAB15489.1 GI:10439338 -   Genbank record update date: Sep. 11, 2006 11:24 PM     Cross References -   Wilson et al (1991) J. Exp. Med. 173:137-146; 30 WO2003/072036     (Claim 1; FIG. 1); IM:107266; NP_001762.1; NM_001771_1.     (27a) CD22 (CD22 Molecule)     Nucleotide -   Genbank accession no X52785 -   Genbank version no. X52785.1 GI:29778 -   Genbank record update date: Feb. 2, 2011 10:09 AM     Polypeptide -   Genbank accession no. CAA36988 -   Genbank version no. CAA36988.1 GI:29779 -   Genbank record update date: Feb. 2, 2011 10:09 AM     Cross References -   Stamenkovic I. et al., Nature 345 (6270), 74-77 (1990)??     Other Information -   Official Symbol: CD22 -   Other Aliases: SIGLEC-2, SIGLEC2 -   Other Designations: B-cell receptor CD22; B-lymphocyte cell adhesion     molecule; BL-CAM; CD22 antigen; T-cell surface antigen Leu-14;     sialic acid binding Ig-like lectin 2; sialic acid-binding Ig-like     lectin 2     Antibodies -   G5/44 (Inotuzumab): DiJoseph J F., et al Cancer Immunol Immunother.     2005 January; 54(1):11-24. -   Epratuzumab—Goldenberg D M., et al Expert Rev Anticancer Ther.     6(10): 1341-53, 2006.     (28) CD79a (CD79A, CD79alpha), Immunoglobulin-Associated Alpha, a B     Cell-Specific Protein that Covalently Interacts with Ig Beta (CD79B)     and Forms a Complex on the Surface with Ig M     35 Molecules, Transduces a Signal Involved in B-Cell     Differentiation), Pl: 4.84, MW: 25028 TM: 2     [P] Gene Chromosome: 19q13.2).     Nucleotide -   Genbank accession no NM_001783 -   Genbank version no. NM_001783.3 GI:90193587 -   Genbank record update date: Jun. 26, 2012 01:48 PM     Polypeptide -   Genbank accession no. NP_001774 -   Genbank version no. NP_001774.1 GI:4502685 -   Genbank record update date: Jun. 26, 2012 01:48 PM     Cross References -   WO2003/088808, US2003/0228319; WO2003/062401 (claim 9);     US2002/150573 (claim 4, pages 13-14); WO99/58658 (claim 13, FIG.     16); WO92/07574 (FIG. 1); U.S. Pat. No. 5,644,033; Ha et     al (1992) J. Immunol. 148(5):1526-1531; Müller et al (1992) Eur. J.     Immunol. 22:1621-1625; Hashimoto et al (1994) Immunogenetics     40(4):287-295; Preud'homme et al (1992) Clin. Exp. 5 Immunol.     90(1):141-146; Yu et al (1992) J. Immunol. 148(2) 633-637; Sakaguchi     et al (1988) -   EMBO J. 7(11):3457-3464     (29) CXCR5 (Burkitt's Lymphoma Receptor 1, a G Protein-Coupled     Receptor that is Activated by the CXCL13 Chemokine, Functions in     Lymphocyte Migration and Humoral Defense, Plays a 10 Role in HIV-2     Infection and Perhaps Development of AIDS, Lymphoma, Myeloma, and     Leukemia); 372 aa, pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome:     11q23.3,     Nucleotide -   Genbank accession no NM_001716 -   Genbank version no. NM_001716.4 GI:342307092 -   Genbank record update date: Sep. 30, 2012 01:49 PM     Polypeptide -   Genbank accession no. NP_001707 -   Genbank version no. NP_001707.1 GI:4502415 -   Genbank record update date: Sep. 30, 2012 01:49 PM     Cross References -   WO2004/040000; WO2004/015426; US2003/105292 (Example 2); U.S. Pat.     No. 6,555,339 (Example 2); WO2002/61087 (FIG. 1); WO2001/57188     (Claim 20, page 269); WO2001/72830 (pages 12-13); WO2000/22129     (Example 1, pages 152-153, 15 Example 2, pages 254-256); WO99/28468     (claim 1, page 38); U.S. Pat. No. 5,440,021 (Example 2, col 49-52);     WO94/28931 (pages 56-58); WO92/17497 (claim 7, FIG. 5); Dobner et     al (1992) Eur. J. Immunol. 22:2795-2799; Barella et al (1995)     Biochem. J. 309:773-779     (30) HLA-DOB (Beta Subunit of MHC Class II Molecule (La Antigen)     that Binds Peptides and 20 Presents them to CD4+ T Lymphocytes); 273     aa, pl: 6.56, MW: 30820. TM: 1 [P] Gene Chromosome: 6p21.3)     Nucleotide -   Genbank accession no NM_002120 -   Genbank version no. NM_002120.3 GI:118402587 -   Genbank record update date: Sep. 8, 2012 04:46 PM     Polypeptide -   Genbank accession no. NP_002111 -   Genbank version no. NP_002111.1 GI:4504403 -   Genbank record update date: Sep. 8, 2012 04:46 PM     Cross References -   Tonnelle et al (1985) EMBO J. 4(11):2839-2847; Jonsson et al (1989)     Immunogenetics 29(6):411-413; Beck et al (1992) J. Mol. Biol.     228:433-441; Strausberg et al (2002) Proc. Natl. Acad. Sci USA     99:16899-16903; Servenius et al (1987) J. Biol. Chem. 262:8759-8766;     Beck et al (1996) J. Mol. Biol. 25 255:1-13; Naruse et al (2002)     Tissue Antigens 59:512-519; WO99/58658 (claim 13, FIG. 15); U.S.     Pat. No. 6,153,408 (Col 35-38); U.S. Pat. No. 5,976,551 (col     168-170); U.S. Pat. No. 6,011,146 (col 145-146); Kasahara et     al (1989) Immunogenetics 30(1):66-68; Larhammar et al (1985) J.     Biol. Chem. 260(26):14111-14119     (31) P2X5 (Purinergic Receptor P2X Ligand-Gated Ion Channel 5, an     Ion Channel Gated by Extracellular ATP, May be Involved in Synaptic     Transmission and Neurogenesis, Deficiency May Contribute to the     Pathophysiology of Idiopathic Detrusor Instability); 422 aa), pl:     7.63, MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3).     Nucleotide -   Genbank accession no NM_002561 -   Genbank version no. NM_002561.3 GI:325197202 -   Genbank record update date: Jun. 27, 2012 12:41 AM     Polypeptide -   Genbank accession no. NP_002552 -   Genbank version no. NP_002552.2 GI:28416933 -   Genbank record update date: Jun. 27, 2012 12:41 AM     Cross References -   Le et al (1997) FEBS Lett. 418(1-2):195-199; WO2004/047749;     WO2003/072035 (claim 10); Touchman et al (2000) Genome Res.     10:165-173; WO2002/22660 (claim 20); WO2003/093444 (claim 1);     WO2003/087768 (claim 1); WO2003/029277 (page 82)     (32) CD72 (B-Cell Differentiation Antigen CD72, Lyb-2); 359 aa, pl:     8.66, MW: 40225, TM: 1 5 [P] Gene Chromosome: 9p13.3).     Nucleotide -   Genbank accession no NM_001782 -   Genbank version no. NM_001782.2 GI:194018444 -   Genbank record update date: Jun. 26, 2012 01:43 PM     Polypeptide -   Genbank accession no. NP_001773 -   Genbank version no. NP_001773.1 GI:4502683 -   Genbank record update date: Jun. 26, 2012 01:43 PM     Cross References -   WO2004042346 (claim 65); WO2003/026493 (pages 51-52, 57-58);     WO2000/75655 (pages 105-106); Von Hoegen et al (1990) J. Immunol.     144(12):4870-4877; Strausberg et al (2002) Proc. Natl. Acad. Sci USA     99:16899-16903.     (33) LY64 (Lymphocyte Antigen 64 (RP105), Type I Membrane Protein of     the Leucine Rich Repeat (LRR) Family, Regulates B-Cell Activation     and Apoptosis, Loss of Function is Associated with Increased Disease     Activity in Patients with Systemic Lupus Erythematosis); 661 aa, pl:     6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12).     Nucleotide -   Genbank accession no NM_005582 -   Genbank version no. NM_005582.2 GI:167555126 -   Genbank record update date: Sep. 2, 2012 01:50 PM     Polypeptide -   Genbank accession no. NP_005573 -   Genbank version no. NP_005573.2 GI:167555127 -   Genbank record update date: Sep. 2, 2012 01:50 PM     Cross References -   US2002/193567; WO97/07198 (claim 11, pages 39-42); Miura et     al (1996) 15 Genomics 38(3):299-304; Miura et al (1998) Blood     92:2815-2822; WO2003/083047; WO97/44452 (claim 8, pages 57-61);     WO2000/12130 (pages 24-26).     (34) FcRH1 (Fc Receptor-Like Protein 1, a Putative Receptor for the     Immunoglobulin Fc Domain that Contains C2 Type Ig-Like and ITAM     Domains, May have a Role in B-Lymphocyte 20 Differentiation); 429     aa, pl: 5.28, MW: 46925 TM: 1 [P] Gene Chromosome: 1q21-1q22)     Nucleotide -   Genbank accession no NM_052938 -   Genbank version no. NM_052938.4 GI:226958543 -   Genbank record update date: Sep. 2, 2012 01:43 PM     Polypeptide -   Genbank accession no. NP_443170 -   Genbank version no. NP_443170.1 GI:16418419 -   Genbank record update date: Sep. 2, 2012 01:43 PM     Cross References -   WO2003/077836; WO2001/38490 (claim 6, FIG. 18E-1-18-E-2); Davis et     al (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777; WO2003/089624     (claim 8); EP1347046 (claim 1); WO2003/089624 (claim 7).     (35) IRTA2 (Immunoglobulin Superfamily Receptor Translocation     Associated 2, a Putative Immunoreceptor with Possible Roles in B     Cell Development and Lymphomagenesis; Deregulation of the Gene by     Translocation Occurs in Some B Cell Malignancies); 977 aa, pl: 6.88,     MW: 106468, TM: 1 [P] Gene Chromosome: 1q21)     Nucleotide -   Genbank accession no AF343662 -   Genbank version no. AF343662.1 GI:13591709 -   Genbank record update date: Mar. 11, 2010 01:16 AM     Polypeptide -   Genbank accession no. AAK31325 -   Genbank version no. AAK31325.1 GI:13591710 -   Genbank record update date: Mar. 11, 2010 01:16 AM     Cross References -   AF343663, AF343664, AF343665, AF369794, AF397453, AK090423,     AK090475, AL834187, AY358085; Mouse:AK089756, AY158090, AY506558;     NP_112571.1; WO2003/024392 (claim 2, FIG. 97); Nakayama et al (2000)     Biochem. Biophys. Res. Commun. 277(1):124-127; WO2003/077836;     WO2001/38490 (claim 3, FIG. 18B-1-18B-2).     (36) TENB2 (TMEFF2, Tomoregulin, TPEF, HPP1, TR, Putative     Transmembrane 35 Proteoglycan, Related to the EGF/Heregulin Family     of Growth Factors and Follistatin); 374 aa)     Nucleotide -   Genbank accession no AF179274 -   Genbank version no. AF179274.2 GI:12280939 -   Genbank record update date: Mar. 11, 2010 01:05 AM     Polypeptide -   Genbank accession no. AAD55776 -   Genbank version no. AAD55776.2 GI:12280940 -   Genbank record update date: Mar. 11, 2010 01:05 AM     Cross References -   NCBI Accession: AAD55776, AAF91397, AAG49451, NCBI RefSeq:     NP_057276; NCBI Gene: 23671; OMIM: 605734; SwissProt Q9UIK5;     AY358907, CAF85723, CQ782436; WO2004/074320; JP2004113151;     WO2003/042661; WO2003/009814; EP1295944 (pages 69-70); WO2002/30268     (page 329); WO2001/90304; US2004/249130; US2004/022727;     WO2004/063355; US2004/197325; US2003/232350; 5 US2004/005563;     US2003/124579; Horie et al (2000) Genomics 67:146-152; Uchida et     al (1999) Biochem. Biophys. Res. Commun. 266:593-602; Liang et     al (2000) Cancer Res. 60:4907-12; Glynne-Jones et al (2001) Int J     Cancer. October 15; 94(2):178-84. (37) PSMA—FOLH1 (Folate Hydrolase     (Prostate-Specific Membrane Antigen) 1)     Nucleotide -   Genbank accession no M99487 -   Genbank version no. M99487.1 GI:190663 -   Genbank record update date: Jun. 23, 2010 08:48 AM     Polypeptide -   Genbank accession no. AAA60209 -   Genbank version no. AAA60209.1 GI:190664 -   Genbank record update date: Jun. 23, 2010 08:48 AM     Cross References -   Israeli R. S., et al Cancer Res. 53 (2), 227-230 (1993)     Other Information -   Official Symbol: FOLH1 -   Other Aliases: GIG27, FGCP, FOLH, GCP2, GCPII, NAALAD1, NAALAdase,     PSM, PSMA, mGCP -   Other Designations: N-acetylated alpha-linked acidic dipeptidase 1;     N-acetylated-alpha-linked acidic dipeptidase I; NAALADase I; cell     growth-inhibiting gene 27 protein; folylpoly-gamma-glutamate     carboxypeptidase; glutamate carboxylase II; glutamate     carboxypeptidase 2; glutamate carboxypeptidase II; membrane     glutamate carboxypeptidase; prostate specific membrane antigen     variant F; pteroylpoly-gamma-glutamate carboxypeptidase     Antibodies -   U.S. Pat. No. 7,666,425: -   Antibodies produces by Hybridomas having the following ATCC     references: ATCC accession No. HB-12101, ATCC accession No.     HB-12109, ATCC accession No. HB-12127 and ATCC accession No.     HB-12126. -   Proscan: a monoclonal antibody selected from the group consisting of     8H12, 3E11, 17G1, 29B4, 30C1 and 20F2 (U.S. Pat. No. 7,811,564;     Moffett S., et al Hybridoma (Larchmt). 2007 Dec.; 26(6):363-72). -   Cytogen: monoclonal antibodies 7E11-C5 (ATCC accession No. HB 10494)     and 9H10-A4 (ATCC accession No. HB11430)—U.S. Pat. No. 5,763,202 -   GlycoMimetics: NUH2—ATCC accession No. HB 9762 (U.S. Pat. No.     7,135,301) -   Human Genome Science: HPRAJ70—ATCC accession No. 97131 (U.S. Pat.     No. 6,824,993); Amino acid sequence encoded by the cDNA clone     (HPRAJ70) deposited as American Type Culture Collection (“ATCC”)     Deposit No. 97131 -   Medarex: Anti-PSMA antibodies that lack fucosyl residues—U.S. Pat.     No. 7,875,278

Mouse anti-PSMA antibodies include the 3F5.4G6, 3D7.1.1, 4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6, 4C8B9, and monoclonal antibodies. Hybridomas secreting 3F5.4G6, 3D7.1.1, 4E10-1.14, 3E11, 4D8, 3E6, 3C9, 2C7, 1G3, 3C4, 3C6, 4D4, 1G9, 5C8B9, 3G6 or 4C8B9 have been publicly deposited and are described in U.S. Pat. No. 6,159,508. Relevant hybridomas have been publicly deposited and are described in U.S. Pat. No. 6,107,090. Moreover, humanized anti-PSMA antibodies, including a humanized version of J591, are described in further detail in PCT Publication WO 02/098897.

Other mouse anti-human PSMA antibodies have been described in the art, such as mAb 107-1A4 (Wang, S. et al. (2001) Int. J. Cancer 92:871-876) and mAb 2C9 (Kato, K. et al. (2003) Int. J. Urol. 10:439-444).

Examples of human anti-PSMA monoclonal antibodies include the 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 antibodies, isolated and structurally characterized as originally described in PCT Publications WO 01/09192 and WO 03/064606 and in U.S. Provisional Application Ser. No. 60/654,125, entitled “Human Monoclonal Antibodies to Prostate Specific Membrane Antigen (PSMA)”, filed on Feb. 18, 2005. The V.sub.H amino acid sequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 are shown in SEQ ID NOs: 1-9, respectively. The V.sub.L amino acid sequences of 4A3, 7F12, 8C12, 8A11, 16F9, 2A10, 2C6, 2F5 and 1C3 are shown in SEQ ID NOs: 10-18, respectively.

Other human anti-PSMA antibodies include the antibodies disclosed in PCT Publication WO 03/034903 and US Application No. 2004/0033229.

-   NW Biotherapeutics: A hybridoma cell line selected from the group     consisting of 3F5.4G6 having ATCC accession number HB12060, 3D7-1.I.     having ATCC accession number HB12309, 4E10-1.14 having ATCC     accession number HB12310, 3E11 (ATCC HB12488), 4D8 (ATCC HB12487),     3E6 (ATCC HB12486), 3C9 (ATCC HB12484), 2C7 (ATCC HB12490), 1G3     (ATCC HB12489), 3C4 (ATCC HB12494), 3C6 (ATCC HB12491), 4D4 (ATCC     HB12493), 1G9 (ATCC HB12495), 5C8B9 (ATCC HB12492) and 3G6 (ATCC     HB12485)—see U.S. Pat. No. 6,150,508 -   PSMA Development Company/Progenics/Cytogen—Seattle Genetics: mAb     3.9, produced by the hybridoma deposited under ATCC Accession No.     PTA-3258 or mAb 10.3, produced by the hybridoma deposited under ATCC     Accession No. PTA-3347—U.S. Pat. No. 7,850,971 -   PSMA Development Company—Compositions of PSMA antibodies (US     20080286284, Table 1)     -   This application is a divisional of U.S. patent application Ser.         No. 10/395,894, filed on Mar. 21, 2003 (U.S. Pat. No. 7,850,971) -   University Hospital Freiburg, Germany—mAbs 3/A12, 3/E7, and 3/F11     (Wolf P., et al Prostate. 2010 Apr. 1; 70(5):562-9).     (38) SST (Somatostatin Receptor; Note that there are 5 Subtypes)     (38.1) SSTR2 (Somatostatin Receptor 2)     Nucleotide -   Genbank accession no NM_001050 -   Genbank version no. NM_001050.2 GI:44890054 -   Genbank record update date: Aug. 19, 2012 01:37 PM     Polypeptide -   Genbank accession no. NP_001041 -   Genbank version no. NP_001041.1 GI:4557859 -   Genbank record update date: Aug. 19, 2012 01:37 PM     Cross References -   Yamada Y., et al Proc. Natl. Acad. Sci. U.S.A. 89 (1), 251-255     (1992); Susini C., et al Ann Oncol. 2006 Dec.; 17(12):1733-42     Other Information -   Official Symbol: SSTR2 -   Other Designations: SRIF-1; SS2R; somatostatin receptor type 2     (38.2) SSTR5 (Somatostatin Receptor 5)     Nucleotide -   Genbank accession no D16827 -   Genbank version no. D16827.1 GI:487683 -   Genbank record update date: Aug. 1, 2006 12:45 PM     Polypeptide -   Genbank accession no. BAA04107 -   Genbank version no. BAA04107.1 GI:487684 -   Genbank record update date: Aug. 1, 2006 12:45 PM     Cross References -   Yamada, Y., et al Biochem. Biophys. Res. Commun. 195 (2), 844-852     (1993)     Other Information -   Official Symbol: SSTR5 -   Other Aliases: SS-5-R -   Other Designations: Somatostatin receptor subtype 5; somatostatin     receptor type 5     (38.3) SSTR1     (38.4) SSTR3     (38.5) SSTR4 -   AvB6—Both Subunits (39+40)     (39) ITGAV (Integrin, Alpha V;     Nucleotide -   Genbank accession no M14648 J02826 M18365 -   Genbank version no. M14648.1 GI:340306 -   Genbank record update date: Jun. 23, 2010 08:56 AM     Polypeptide -   Genbank accession no. AAA36808 -   Genbank version no. AAA36808.1 GI:340307 -   Genbank record update date: Jun. 23, 2010 08:56 AM     Cross References -   Suzuki S., et al Proc. Natl. Acad. Sci. U.S.A. 83 (22), 8614-8618     (1986)     Other Information -   Official Symbol: ITGAV -   Other Aliases: CD51, MSK8, VNRA, VTNR -   Other Designations: antigen identified by monoclonal antibody L230;     integrin alpha-V; integrin alphaVbeta3; integrin, alpha V     (vitronectin receptor, alpha polypeptide, antigen CD51); vitronectin     receptor subunit alpha     (40) ITGB6 (Integrin, Beta 6)     Nucleotide -   Genbank accession no NM_000888 -   Genbank version no. NM_000888.3 GI:9966771 -   Genbank record update date: Jun. 27, 2012 12:46 AM     Polypeptide -   Genbank accession no. NP_000879 -   Genbank version no. NP_000879.2 GI:9625002 -   Genbank record update date: Jun. 27, 2012 12:46 AM     Cross References -   Sheppard D. J., et al Biol. Chem. 265 (20), 11502-11507 (1990)     Other Information -   Official Symbol: ITGB6 -   Other Designations: integrin beta-6     Antibodies -   Biogen: U.S. Pat. No. 7,943,742—Hybridoma clones 6.3G9 and 6.8G6     were deposited with the ATCC, accession numbers ATCC PTA-3649 and     -3645, respectively. -   Biogen: U.S. Pat. No. 7,465,449—In some embodiments, the antibody     comprises the same heavy and light chain polypeptide sequences as an     antibody produced by hybridoma 6.1A8, 6.3G9, 6.8G6, 6.2B1, 6.2B10,     6.2A1, 6.2E5, 7.1G10, 7.7G5, or 7.1C5. -   Centocor (J&J): U.S. Pat. No. 7,550,142; U.S. Pat. No. 7,163,681     -   For example in U.S. Pat. No. 7,550,142—an antibody having human         heavy chain and human light chain variable regions comprising         the amino acid sequences shown in SEQ ID NO: 7 and SEQ ID NO: 8. -   Seattle Genetics: 15H3 (Ryan M C., et al Cancer Res Apr. 15, 2012;     72(8 Supplement): 4630)     (41) CEACAM5 (Carcinoembryonic Antigen-Related Cell Adhesion     Molecule 5)     Nucleotide -   Genbank accession no M17303 -   Genbank version no. M17303.1 GI:178676 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Polypeptide -   Genbank accession no. AAB59513 -   Genbank version no. AAB59513.1 GI:178677 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Cross References -   Beauchemin N., et al Mol. Cell. Biol. 7 (9), 3221-3230 (1987)     Other Information -   Official Symbol: CEACAM5 -   Other Aliases: CD66e, CEA -   Other Designations: meconium antigen 100     Antibodies -   AstraZeneca-MedImmune: US 20100330103; US20080057063; US20020142359     -   for example an antibody having complementarity determining         regions (CDRs) with the following sequences: heavy chain;         CDR1—DNYMH, CDR2—WIDPENGDTE YAPKFRG, CDR3—LIYAGYLAMD Y; and         light chain CDR1—SASSSVTYMH, CDR2—STSNLAS, CDR3—QQRSTYPLT.     -   Hybridoma 806.077 deposited as European Collection of Cell         Cultures (ECACC) deposit no. 96022936. -   Research Corporation Technologies, Inc.: U.S. Pat. No. 5,047,507 -   Bayer Corporation: U.S. Pat. No. 6,013,772 -   BioAlliance: U.S. Pat. No. 7,982,017; U.S. Pat. No. 7,674,605     -   U.S. Pat. No. 7,674,605         -   an antibody comprising the heavy chain variable region             sequence from the amino acid sequence of SEQ ID NO: 1, and             the light chain variable region sequence from the amino acid             sequence of SEQ ID NO:2.         -   an antibody comprising the heavy chain variable region             sequence from the amino acid sequence of SEQ ID NO:5, and             the light chain variable region sequence from the amino acid             sequence of SEQ ID NO:6. -   Celltech Therapeutics Limited: U.S. Pat. No. 5,877,293 -   The Dow Chemical Company: U.S. Pat. No. 5,472,693; U.S. Pat. No.     6,417,337; U.S. Pat. No. 6,333,405     -   U.S. Pat. No. 5,472,693—for example, ATCC No. CRL-11215     -   U.S. Pat. No. 6,417,337—for example, ATCC CRL-12208     -   U.S. Pat. No. 6,333,405—for example, ATCC CRL-12208 -   Immunomedics, Inc: U.S. Pat. No. 7,534,431; U.S. Pat. No. 7,230,084;     U.S. Pat. No. 7,300,644; U.S. Pat. No. 6,730,300;     -   US20110189085         -   an antibody having CDRs of the light chain variable region             comprise: CDR1 comprises KASQDVGTSVA (SEQ ID NO: 20); CDR2             comprises WTSTRHT (SEQ ID NO: 21); and CDR3 comprises             QQYSLYRS (SEQ ID NO: 22);         -   and the CDRs of the heavy chain variable region of said             anti-CEA antibody comprise: CDR1 comprises TYWMS (SEQ ID NO:             23); CDR2 comprises EIHPDSSTINYAPSLKD (SEQ ID NO: 24); and             CDR3 comprises LYFGFPWFAY (SEQ ID NO: 25).     -   US20100221175; US20090092598; US20070202044; US20110064653;         US20090185974; US20080069775.         (42) MET (Met Proto-Oncogene; Hepatocyte Growth Factor Receptor)         Nucleotide -   Genbank accession no M35073 -   Genbank version no. M35073.1 GI:187553 -   Genbank record update date: Mar. 6, 2012 11:12 AM     Polypeptide -   Genbank accession no. AAA59589 -   Genbank version no. AAA59589.1 GI:553531 -   Genbank record update date: Mar. 6, 2012 11:12 AM     Cross References -   Dean M., et al Nature 318 (6044), 385-388 (1985)     Other Information -   Official Symbol: MET -   Other Aliases: AUTS9, HGFR, RCCP2, c-Met -   Other Designations: HGF receptor; HGF/SF receptor; SF receptor;     hepatocyte growth factor receptor; met proto-oncogene tyrosine     kinase; proto-oncogene c-Met; scatter factor receptor;     tyrosine-protein kinase Met     Antibodies -   Abgenix/Pfizer: US20100040629     -   for example, the antibody produced by hybridoma 13.3.2 having         American Type Culture Collection (ATCC) accession number         PTA-5026; the antibody produced by hybridoma 9.1.2 having ATCC         accession number PTA-5027; the antibody produced by hybridoma         8.70.2 having ATCC accession number PTA-5028; or the antibody         produced by hybridoma 6.90.3 having ATCC accession number         PTA-5029. -   Amgen/Pfizer: US20050054019     -   for example, an antibody comprising a heavy chain having the         amino acid sequences set forth in SEQ ID NO: 2 where X2 is         glutamate and X4 is serine and a light chain having the amino         acid sequence set forth in SEQ ID NO: 4 where X8 is alanine,         without the signal sequences; an antibody comprising a heavy         chain having the amino acid sequences set forth in SEQ ID NO: 6         and a light chain having the amino acid sequence set forth in         SEQ ID NO: 8, without the signal sequences; an antibody         comprising a heavy chain having the amino acid sequences set         forth in SEQ ID NO: 10 and a light chain having the amino acid         sequence set forth in SEQ ID NO: 12, without the signal         sequences; or an antibody comprising a heavy chain having the         amino acid sequences set forth in SEQ ID NO: 14 and a light         chain having the amino acid sequence set forth in SEQ ID NO: 16,         without the signal sequences. -   Agouron Pharmaceuticals (Now Pfizer): US20060035907 -   Eli Lilly: US20100129369 -   Genentech: U.S. Pat. No. 5,686,292; US20100028337; US20100016241;     US20070129301; US20070098707; US20070092520, US20060270594;     US20060134104; US20060035278; US20050233960; US20050037431     -   U.S. Pat. No. 5,686,292—for example, ATCC HB-11894 and ATCC         HB-11895     -   US 20100016241—for example, ATCC HB-11894 (hybridoma 1A3.3.13)         or HB-11895 (hybridoma 5D5.11.6) -   National Defense Medical Center, Taiwan: Lu R M., et al     Biomaterials. 2011 April; 32(12):3265-74. -   Novartis: US20090175860     -   for example, an antibody comprising the sequences of CDR1, CDR2         and CDR3 of heavy chain 4687, wherein the sequences of CDR1,         CDR2, and CDR3 of heavy chain 4687 are residues 26-35, 50-65,         and 98-102, respectively, of SEQ ID NO: 58; and the sequences of         CDR1, CDR2, and CDR3 of light chain 5097, wherein the sequences         of CDR1, CDR2, and CDR3 of light chain 5097 are residues 24-39,         55-61, and 94-100 of SEQ ID NO: 37. -   Pharmacia Corporation: US20040166544 -   Pierre Fabre: US20110239316, US20110097262, US20100115639 -   Sumsung: US 20110129481—for example a monoclonal antibody produced     from a hybridoma cell having accession number KCLRF-BP-00219 or     accession number of KCLRF-BP-00223. -   Samsung: US 20110104176—for example an antibody produced by a     hybridoma cell having Accession Number: KCLRF-BP-00220. -   University of Turin Medical School: DN-30 Pacchiana G., et al J Biol     Chem. 2010 Nov. 12; 285(46):36149-57 -   Van Andel Research Institute: Jiao Y., et al Mol Biotechnol. 2005     Sep.; 31(1):41-54.     (43) MUC1 (Mucin 1, Cell Surface Associated)     Nucleotide -   Genbank accession no J05581 -   Genbank version no. J05581.1 GI:188869 -   Genbank record update date: Jun. 23, 2010 08:48 AM     Polypeptide -   Genbank accession no. AAA59876 -   Genbank version no. AAA59876.1 GI:188870 -   Genbank record update date: Jun. 23, 2010 08:48 AM     Cross References -   Gendler S. J., et al J. Biol. Chem. 265 (25), 15286-15293 (1990)     Other Information -   Official Symbol: MUC1 -   Other Aliases: RP11-263K19.2, CD227, EMA, H23AG, KL-6, MAM6, MUC-1,     MUC-1/SEC, MUC-1/X, MUC1/ZD, PEM, PEMT, PUM -   Other Designations: DF3 antigen; H23 antigen; breast     carcinoma-associated antigen DF3; carcinoma-associated mucin;     episialin; krebs von den Lungen-6; mucin 1, transmembrane; mucin-1;     peanut-reactive urinary mucin; polymorphic epithelial mucin; tumor     associated epithelial mucin; tumor-associated epithelial membrane     antigen; tumor-associated mucin     Antibodies -   AltaRex—Quest Pharma Tech: U.S. Pat. No. 6,716,966—for example an     Alt-1 antibody produced by the hybridoma ATCC No PTA-975. -   AltaRex—Quest Pharma Tech: U.S. Pat. No. 7,147,850 -   CRT: 5E5—Sorensen A L., et al Glycobiology vol. 16 no. 2 pp. 96-107,     2006; HMFG2—Burchell J., et al Cancer Res., 47, 5476-5482 (1987) -   Glycotope GT-MAB: GT-MAB 2.5-GEX (Website:     http://www.glycotope.com/pipeline/pankomab-gex) -   Immunogen: U.S. Pat. No. 7,202,346     -   for example, antibody MJ-170: hybridoma cell line MJ-170 ATCC         accession no. PTA-5286Monoclonal antibody MJ-171: hybridoma cell         line MJ-171 ATCC accession no. PTA-5287; monoclonal antibody         MJ-172: hybridoma cell line MJ-172 ATCC accession no. PTA-5288;         or monoclonal antibody MJ-173: hybridoma cell line MJ-173 ATCC         accession no. PTA-5302 -   Immunomedics: U.S. Pat. No. 6,653,104 -   Ramot Tel Aviv Uni: U.S. Pat. No. 7,897,351 -   Regents Uni. CA: U.S. Pat. No. 7,183,388; US20040005647;     US20030077676. -   Roche GlycArt: U.S. Pat. No. 8,021,856 -   Russian National Cancer Research Center: Imuteran—Ivanov P K., et al     Biotechnol J. 2007 Jul.; 2(7):863-70 -   Technische Univ Braunschweig: (IIB6, HT186-B7, HT186-D11, HT186-G2,     HT200-3A-C1, HT220-M-D1, HT220-M-G8)—Thie H., et al PLoS One. 2011     Jan. 14; 6(1):e15921     (44) CA9 (Carbonic Anhydrase IX)     Nucleotide -   Genbank accession no. X66839 -   Genbank version no. X66839.1 GI:1000701 -   Genbank record update date: Feb. 2, 2011 10:15 AM     Polypeptide -   Genbank accession no. CAA47315 -   Genbank version no. CAA47315.1 GI:1000702 -   Genbank record update date: Feb. 2, 2011 10:15 AM     Cross References -   Pastorek J., et al Oncogene 9 (10), 2877-2888 (1994)     Other Information -   Official Symbol: CA9 -   Other Aliases: CAIX, MN -   Other Designations: CA-IX; P54/58N; RCC-associated antigen G250;     RCC-associated protein G250; carbonate dehydratase IX; carbonic     anhydrase 9; carbonic dehydratase; membrane antigen MN; pMW1; renal     cell carcinoma-associated antigen G250     Antibodies -   Abgenix/Amgen: US20040018198 -   Affibody: Anti-CAIX Affibody molecules     (http://www.affibody.com/en/Product-Portfolio/Pipeline/) -   Bayer: U.S. Pat. No. 7,462,696 -   Bayer/Morphosys: 3ee9 mAb—Petrul H M., et al Mol Cancer Ther. 2012     Feb.; 11(2):340-9 -   Harvard Medical School: Antibodies G10, G36, G37, G39, G45, G57,     G106, G119, G6, G27, G40 and G125. Xu C., et al PLoS One. 2010 Mar.     10; 5(3):e9625 -   Institute of Virology, Slovak Academy of Sciences (Bayer)—U.S. Pat.     No. 5,955,075     -   for example, M75—ATCC Accession No. HB 11128 or MN12—ATCC         Accession No. HB 11647 -   Institute of Virology, Slovak Academy of Sciences: U.S. Pat. No.     7,816,493     -   for example the M75 monoclonal antibody that is secreted from         the hybridoma VU-M75, which was deposited at the American Type         Culture Collection under ATCC No. HB 11128; or the V/10         monoclonal antibody secreted from the hybridoma V/10-VU, which         was deposited at the International Depository Authority of the         Belgian Coordinated Collection of Microorganisms (BCCM) at the         Laboratorium voor Moleculaire Bioloqie-Plasmidencollectie (LMBP)         at the Universeit Gent in Gent, Belgium, under Accession No.         LMBP 6009CB. -   Institute of Virology, Slovak Academy of Sciences US20080177046;     US20080176310; US20080176258; US20050031623 -   Novartis: US20090252738 -   Wilex: U.S. Pat. No. 7,691,375—for example the antibody produced by     the hybridoma cell line DSM ASC 2526. -   Wilex: US20110123537; Rencarex: Kennett R H., et al Curr Opin Mol     Ther. 2003 Feb.; 5(1):70-5 -   Xencor: US20090162382     (45) EGFRv/II (Epidermal Growth Factor Receptor (EGFR), Transcript     Variant 3,     Nucleotide -   Genbank accession no. NM_201283 -   Genbank version no. NM_201283.1 GI:41327733 -   Genbank record update date: Sep. 30, 2012 01:47 PM     Polypeptide -   Genbank accession no. NP_958440 -   Genbank version no. NP_958440.1 GI:41327734 -   Genbank record update date: Sep. 30, 2012 01:47 PM     Cross-References -   Batra S K., et al Cell Growth Differ 1995; 6:1251-1259.     Antibodies: -   U.S. Pat. No. 7,628,986 and U.S. Pat. No. 7,736,644 (Amgen)     -   For example, a heavy chain variable region amino acid sequence         selected from the group consisting of SEQ ID NO: 142 and         variants & a light chain variable region amino acid sequence         selected from the group consisting of: SEQ ID NO: 144 and         variants. -   US20100111979 (Amgen)     -   For example, an antibody comprising a heavy chain amino acid         sequence comprising:     -   CDR1 consisting of a sequence selected from the group consisting         of the amino acid sequences for the CDR1 region of antibodies         13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),         150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139         (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318         (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);     -   CDR2 consisting of a sequence selected from the group consisting         of the amino acid sequences for the CDR2 region of antibodies         13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),         150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139         (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318         (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17);         and     -   CDR3 consisting of a sequence selected from the group consisting         of the amino acid sequences for the CDR3 region of antibodies         13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO: 2), 170 (SEQ ID NO: 4),         150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7), 250 (SEQ ID NO: 9), 139         (SEQ ID NO: 10), 211 (SEQ ID NO: 12), 124 (SEQ ID NO: 13), 318         (SEQ ID NO: 15), 342 (SEQ ID NO: 16), and 333 (SEQ ID NO: 17). -   US20090240038 (Amgen)     -   For example, an antibody having at least one of the heavy or         light chain polypeptides comprises an amino acid sequence that         is at least 90% identical to the amino acid sequence selected         from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ         ID NO: 142, SEQ ID NO: 144, and any combination thereof. -   US20090175887 (Amgen)     -   For example, an antibody having a heavy chain amino acid         sequence selected from the group consisting of the heavy chain         amino acid sequence of antibody 13.1.2 (SEQ ID NO: 138), 131         (SEQ ID NO: 2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ         ID NO: 7), 250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID         NO: 12), 124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID         NO: 16), and 333 (SEQ ID NO: 17). -   US20090156790 (Amgen)     -   For example, antibody having heavy chain polypeptide and a light         chain polypeptide, wherein at least one of the heavy or light         chain polypeptides comprises an amino acid sequence that is at         least 90% identical to the amino acid sequence selected from the         group consisting of: SEQ ID NO: 2, SEQ ID NO: 19, SEQ ID NO:         142, SEQ ID NO: 144, and any combination thereof. -   US20090155282, US20050059087 and US20050053608 (Amgen)     -   For example, an antibody heavy chain amino acid sequence         selected from the group consisting of the heavy chain amino acid         sequence of antibody 13.1.2 (SEQ ID NO: 138), 131 (SEQ ID NO:         2), 170 (SEQ ID NO: 4), 150 (SEQ ID NO: 5), 095 (SEQ ID NO: 7),         250 (SEQ ID NO: 9), 139 (SEQ ID NO: 10), 211 (SEQ ID NO: 12),         124 (SEQ ID NO: 13), 318 (SEQ ID NO: 15), 342 (SEQ ID NO: 16),         and 333 (SEQ ID NO: 17). -   MR1-1 (U.S. Pat. No. 7,129,332; Duke)     -   For example, a variant antibody having the sequence of SEQ ID         NO. 18 with the substitutions S98P-T99Y in the CDR3 VH, and F92W         in CDR3 VL. -   L8A4, H10, Y10 (Wikstrand C J., et al Cancer Res. 1995 Jul. 15;     55(14):3140-8; Duke) -   US20090311803 (Harvard University)     -   For example, SEQ ID NO:9 for antibody heavy chain variable         region, and SEQ ID NO: 3 for light chain variable region amino         acid sequences US20070274991 (EMD72000, also known as matuzumab;         Harvard University)     -   For example, SEQ ID NOs: 3 & 9 for light chain and heavy chain         respectively -   U.S. Pat. No. 6,129,915 (Schering)     -   For example, SEQ. ID NOs: 1, 2, 3, 4, 5 and 6. -   mAb CH12—Wang H., et al FASEB J. 2012 Jan.; 26(1):73-80 (Shanghai     Cancer Institute). -   RAbDMvIII—Gupta P., et al BMC Biotechnol. 2010 Oct. 7; 10:72     (Stanford University Medical Center). -   mAb Ua30—Ohman L., et al Tumour Biol. 2002 Mar.-Apr.; 23(2):61-9     (Uppsala University). -   Han D G., et al Nan Fang Yi Ke Da Xue Xue Bao. 2010 Jan.; 30(1):25-9     (Xi'an Jiaotong University).     (46) CD33 (CD33 Molecule)     Nucleotide -   Genbank accession no. M_23197 -   Genbank version no. NM_23197.1 GI:180097 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Polypeptide -   Genbank accession no. AAA51948 -   Genbank version no. AAA51948.1 GI:188098 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Cross-References -   Simmons D., et al J. Immunol. 141 (8), 2797-2800 (1988)     Other Information -   Official Symbol: CD33 -   Other Aliases: SIGLEC-3, SIGLEC3, p67 -   Other Designations: CD33 antigen (gp67); gp67; myeloid cell surface     antigen CD33; sialic acid binding Ig-like lectin 3; sialic     acid-binding Ig-like lectin     Antibodies -   H195 (Lintuzumab)—Raza A., et al Leuk Lymphoma. 2009 August;     50(8):1336-44; U.S. Pat. No. 6,759,045 (Seattle     Genetics/Immunomedics) -   mAb OKT9: Sutherland, D. R. et al. Proc NatlAcad Sci USA 78(7):     4515-4519 1981, Schneider, C., et al J Biol Chem 257, 8516-8522     (1982) -   mAb E6: Hoogenboom, H. R., et al J Immunol 144, 3211-3217 (1990) -   U.S. Pat. No. 6,590,088 (Human Genome Sciences)     -   For example, SEQ ID NOs: 1 and 2 and ATCC accession no. 97521 -   U.S. Pat. No. 7,557,189 (Immunogen)     -   For example, an antibody or fragment thereof comprising a heavy         chain variable region which comprises three CDRs having the         amino acid sequences of SEQ ID NOs:1-3 and a light chain         variable region comprising three CDRs having the amino acid         sequences of SEQ ID NOs:4-6.         (47) CD19 (CD19 Molecule)         Nucleotide -   Genbank accession no. NM_001178098 -   Genbank version no. NM_001178098.1 GI:296010920 -   Genbank record update date: Sep. 10, 2012 12:43 AM     Polypeptide -   Genbank accession no. NP_001171569 -   Genbank version no. NP_001171569.1 GI:296010921 -   Genbank record update date: Sep. 10, 2012 12:43 AM     Cross-References -   Tedder T F., et al J. Immunol. 143 (2): 712-7 (1989)     Other Information -   Official Symbol: CD19 -   Other Aliases: B4, CVID3 -   Other Designations: B-lymphocyte antigen CD19; B-lymphocyte surface     antigen B4; T-cell surface antigen Leu-12; differentiation antigen     CD19     Antibodies -   Immunogen: HuB4—Al-Katib A M., et al Clin Cancer Res. 2009 Jun. 15;     15(12):4038-45. -   4G7: Kügler M., et al Protein Eng Des Sel. 2009 Mar.; 22(3):135-47     -   For example, sequences in FIG. 3 of Knappik, A. et al. J Mol         Biol 2000 February; 296(1):57-86 -   AstraZeneca/MedImmune: MEDI-551—Herbst R., et al J Pharmacol Exp     Ther. 2010 October; 335(1):213-22 -   Glenmark Pharmaceuticals: GBR-401—Hou S., et al Mol Cancer Ther     November 2011 10 (Meeting Abstract Supplement) C164 -   U.S. Pat. No. 7,109,304 (Immunomedics)     -   For example, an antibody comprising the sequence of hA19Vk (SEQ         ID NO:7) and the sequence of hA19VH (SEQ ID NO:10) -   U.S. Pat. No. 7,902,338 (Immunomedics)     -   For example, an antibody or antigen-binding fragment thereof         that comprises the light chain complementarity determining         region CDR sequences CDR1 of SEQ ID NO: 16 (KASQSVDYDGDSYLN);         CDR2 of SEQ ID NO: 17 (DASNLVS); and CDR3 of SEQ ID NO: 18         (QQSTEDPWT) and the heavy chain CDR sequences CDR1 of SEQ ID NO:         19 (SYWMN); CDR2 of SEQ ID NO: 20 (QIWPGDGDTNYNGKFKG) and CDR3         of SEQ ID NO: 21 (RETTTVGRYYYAMDY) and also comprises human         antibody framework (FR) and constant region sequences with one         or more framework region amino acid residues substituted from         the corresponding framework region sequences of the parent         murine antibody, and wherein said substituted FR residues         comprise the substitution of serine for phenylalanine at Kabat         residue 91 of the heavy chain variable region. -   Medarex: MDX-1342—Cardarelli P M., et al Cancer Immunol Immunother.     2010 February; 59(2):257-65. -   MorphoSys/Xencor: MOR-208/XmAb-5574—Zalevsky J., et al Blood. 2009     Apr. 16; 113(16):3735-43 -   U.S. Pat. No. 7,968,687 (Seattle Genetics)     -   An antibody or antigen-binding fragment comprising a heavy chain         variable domain comprising the amino acid sequence of SEQ ID         NO:9 and a light chain variable domain comprising the amino acid         sequence of SEQ ID NO: 24. -   4G7 chim—Lang P., et al Blood. 2004 May 15; 103(10):3982-5     (University of Tübingen)     -   For example, FIG. 6 and SEQ ID No: 80 of US20120082664 -   Zhejiang University School of Medicine: 2E8—Zhang J., et al J Drug     Target. 2010 Nov.; 18(9):675-8     (48) IL2RA (Interleukin 2 Receptor, Alpha); NCBI Reference Sequence:     NM_000417.2);     Nucleotide -   Genbank accession no. NM_000417 -   Genbank version no. NM_000417.2 GI:269973860 -   Genbank record update date: Sep. 9, 2012 04:59 PM     Polypeptide -   Genbank accession no. NP_000408 -   Genbank version no. NP_000408.1 GI:4557667 -   Genbank record update date: Sep. 9, 2012 04:59 PM     Cross-References -   Kuziel W. A., et al J. Invest. Dermatol. 94 (6 SUPPL), 27S-32S     (1990)     Other Information -   Official Symbol: IL2RA -   Other Aliases: RP11-536K7.1, CD25, IDDM10, IL2R, TCGFR -   Other Designations: FIL-2 receptor subunit alpha; IL-2-RA; IL-2R     subunit alpha; IL2-RA; TAC antigen; interleukin-2 receptor subunit     alpha; p55     Antibodies -   U.S. Pat. No. 6,383,487 (Novartis/UCL: Baxilisimab [Simulect]) -   U.S. Pat. No. 6,521,230 (Novartis/UCL: Baxilisimab [Simulect])     -   For example, an antibody having an antigen binding site         comprises at least one domain which comprises CDR1 having the         amino acid sequence in SEQ. ID. NO: 7, CDR2 having the amino         acid sequence in SEQ. ID. NO: 8, and CDR3 having the amino acid         sequence in SEQ. ID. NO: 9; or said CDR1, CDR2 and CDR3 taken in         sequence as a whole comprise an amino acid sequence which is at         least 90% identical to SEQ. ID. NOs: 7, 8 and 9 taken in         sequence as a whole. -   Daclizumab—Rech A J., et al Ann NY Acad Sci. 2009 September;     1174:99-106 (Roche)     (49) AXL (AXL Receptor Tyrosine Kinase)     Nucleotide -   Genbank accession no. M76125 -   Genbank version no. M76125.1 GI:292869 -   Genbank record update date: Jun. 23, 2010 08:53 AM     Polypeptide -   Genbank accession no. AAA61243 -   Genbank version no. AAA61243.1 GI:29870 -   Genbank record update date: Jun. 23, 2010 08:53 AM     Cross-References -   O'Bryan J. P., et al Mol. Cell. Biol. 11 (10), 5016-5031 (1991);     Bergsagel P. L., et al J. Immunol. 148 (2), 590-596 (1992)     Other Information -   Official Symbol: AXL -   Other Aliases: JTK11, UFO -   Other Designations: AXL oncogene; AXL transforming sequence/gene;     oncogene AXL; tyrosine-protein kinase receptor UFO     Antibodies -   YW327.6S2—Ye X., et al Oncogene. 2010 Sep. 23; 29(38):5254-64.     (Genentech) -   BergenBio: BGB324 (http://www.bergenbio.com/BGB324)     (50) CD30—TNFRSF8 (Tumor Necrosis Factor Receptor Superfamily,     Member 8)     Nucleotide -   Genbank accession no. M83554 -   Genbank version no. M83554.1 GI:180095 -   Genbank record update date: Jun. 23, 2010 08:53 AM     Polypeptide -   Genbank accession no. AAA51947 -   Genbank version no. AAA51947.1 GI:180096 -   Genbank record update date: Jun. 23, 2010 08:53 AM     Cross-References -   Durkop H., et al Cell 68 (3), 421-427 (1992)     Other Information -   Official Symbol: TNFRSF8 -   Other Aliases: CD30, D1S166E, Ki-1 -   Other Designations: CD30L receptor; Ki-1 antigen; cytokine receptor     CD30; lymphocyte activation antigen CD30; tumor necrosis factor     receptor superfamily member 8     (51) BCMA (B-Cell Maturation Antigen)—TNFRSF17 (Tumor Necrosis     Factor Receptor Superfamily, Member 17)     Nucleotide -   Genbank accession no. Z29574 -   Genbank version no. Z29574.1 GI:471244 -   Genbank record update date: Feb. 2, 2011 10:40 AM     Polypeptide -   Genbank accession no. CAA82690 -   Genbank version no. CAA82690.1 GI:471245 -   Genbank record update date: Feb. 2, 2011 10:40 AM     Cross-References -   Laabi Y., et al Nucleic Acids Res. 22 (7), 1147-1154 (1994)     Other Information -   Official Symbol: TNFRSF17 -   Other Aliases: BCM, BCMA, CD269 -   Other Designations: B cell maturation antigen; B-cell maturation     factor; B-cell maturation protein; tumor necrosis factor receptor     superfamily member 17     (52) CT Ags—CTA (Cancer Testis Antigens)     Cross-References -   Fratta E., et al. Mol Oncol. 2011 Apr.; 5(2):164-82; Lim S H., at al     Am J Blood Res. 2012; 2(1):29-35.     (53) CD174 (Lewis Y)—FUT3 (Fucosyltransferase 3 (Galactoside     3(4)-L-Fucosyltransferase, Lewis Blood Group)     Nucleotide -   Genbank accession no. NM000149 -   Genbank version no. NM000149.3 GI:148277008 -   Genbank record update date: Jun. 26, 2012 04:49 PM     Polypeptide -   Genbank accession no. NP_000140 -   Genbank version no. NP_000140.1 GI:4503809 -   Genbank record update date: Jun. 26, 2012 04:49 PM     Cross-References -   Kukowska-Latallo, J. F., et al Genes Dev. 4 (8), 1288-1303 (1990)     Other Information -   Official Symbol: FUT3 -   Other Aliases: CD174, FT3B, FucT-III, LE, Les -   Other Designations: Lewis F T; alpha-(1,3/1,4)-fucosyltransferase;     blood group Lewis alpha-4-fucosyltransferase; fucosyltransferase     Ill; galactoside 3(4)-L-fucosyltransferase     (54) CLEC14A (C-Type Lectin Domain Family 14, Member A; Genbank     Accession No. NM175060)     Nucleotide -   Genbank accession no. NM175060 -   Genbank version no. NM175060.2 GI:371123930 -   Genbank record update date: Apr. 1, 2012 03:34 PM     Polypeptide -   Genbank accession no. NP_778230 -   Genbank version no. NP_778230.1 GI:28269707 -   Genbank record update date: Apr. 1, 2012 03:34 PM     Other Information -   Official Symbol: CLEC14A -   Other Aliases: UNQ236/PRO269, C14orf27, CEG1, EGFR-5 -   Other Designations: C-type lectin domain family 14 member A; CIECT     and EGF-like domain containing protein; epidermal growth factor     receptor 5     (55) GRP78—HSPA5 (Heat Shock 70 kDa Protein 5 (Glucose-Regulated     Protein, 78 kDa)     Nucleotide -   Genbank accession no. NM005347 -   Genbank version no. NM005347.4 GI:305855105 -   Genbank record update date: Sep. 30, 2012 01:42 PM     Polypeptide -   Genbank accession no. NP_005338 -   Genbank version no. NP_005338.1 GI:16507237 -   Genbank record update date: Sep. 30, 2012 01:42 PM     Cross-References -   Ting J., et al DNA 7 (4), 275-286 (1988) Other infromation -   Official Symbol: HSPA5 -   Other Aliases: BIP, GRP78, MIF2 -   Other Designations: 78 kDa glucose-regulated protein; endoplasmic     reticulum lumenal Ca(2+)-binding protein grp78; immunoglobulin heavy     chain-binding protein     (56) CD70 (CD70 Molecule) L08096     Nucleotide -   Genbank accession no. L08096 -   Genbank version no. L08096.1 GI:307127 -   Genbank record update date: Jun. 23, 2012 08:54 AM     Polypeptide -   Genbank accession no. AAA36175 -   Genbank version no. AAA36175.1 GI:307128 -   Genbank record update date: Jun. 23, 2012 08:54 AM     Cross-References -   Goodwin R. G., et al Cell 73 (3), 447-456 (1993)     Other Information -   Official Symbol: CD70 -   Other Aliases: CD27L, CD27LG, TNFSF7 -   Other Designations: CD27 ligand; CD27-L; CD70 antigen; Ki-24     antigen; surface antigen CD70; tumor necrosis factor (ligand)     superfamily, member 7; tumor necrosis factor ligand superfamily     member 7     Antibodies -   MDX-1411 against CD70 (Medarex) -   h1F6 (Oflazoglu, E., et al, Clin Cancer Res. 2008 Oct. 1;     14(19):6171-80; Seattle Genetics)     -   For example, see US20060083736 SEQ ID NOs: 1, 2, 11 and 12 and         FIG. 1.         (57) Stem Cell Specific Antigens. For Example:     -   5T4 (see entry (63) below)     -   CD25 (see entry (48) above)     -   CD32         -   Polypeptide             -   Genbank accession no. ABK42161             -   Genbank version no. ABK42161.1 GI:117616286             -   Genbank record update date: Jul. 25, 2007 03:00 PM     -   LGR5/GPR49         -   Nucleotide             -   Genbank accession no. NM_003667             -   Genbank version no. NM_003667.2 GI:24475886             -   Genbank record update date: Jul. 22, 2012 03:38 PM         -   Polypeptide             -   Genbank accession no. NP_003658             -   Genbank version no. NP_003658.1 GI:4504379             -   Genbank record update date: Jul. 22, 2012 03:38 PM     -   Prominin/CD133         -   Nucleotide             -   Genbank accession no. NM_006017             -   Genbank version no. NM_006017.2 GI:224994187             -   Genbank record update date: Sep. 30, 2012 01:47 PM         -   Polypeptide             -   Genbank accession no. NP_006008             -   Genbank version no. NP_006008.1 GI:5174387             -   Genbank record update date: Sep. 30, 2012 01:47 PM                 (58) ASG-5                 Cross-References -   (Smith L. M., et. al AACR 2010 Annual Meeting (abstract #2590);     Gudas J. M., et. al. AACR 2010 Annual Meeting (abstract #4393)     Antibodies -   Anti-AGS-5 Antibody: M6.131 (Smith, L. M., et. al AACR 2010 Annual     Meeting (abstract #2590)     (59) ENPP3 (Ectonucleotide Pyrophosphatase/Phosphodiesterase 3)     Nucleotide -   Genbank accession no. AF005632 -   Genbank version no. AF005632.2 GI:4432589 -   Genbank record update date: Mar. 10, 2010 09:41 PM     Polypeptide -   Genbank accession no. AAC51813 -   Genbank version no. AAC51813.1 GI:2465540 -   Genbank record update date: Mar. 10, 2010 09:41 PM     Cross-References -   Jin-Hua P., et al Genomics 45 (2), 412-415 (1997)     Other Information -   Official Symbol: ENPP3 -   Other Aliases: RP5-988G15.3, B10, CD203c, NPP3, PD-IBETA, PDNP3 -   Other Designations: E-NPP 3; dJ1005H11.3 (phosphodiesterase     I/nucleotide pyrophosphatase 3); dJ914N13.3 (phosphodiesterase     I/nucleotide pyrophosphatase 3); ectonucleotide     pyrophosphatase/phosphodiesterase family member 3; gp130RB13-6;     phosphodiesterase I beta; phosphodiesterase I/nucleotide     pyrophosphatase 3; phosphodiesterase-I beta     (60) PRR4 (Proline Rich 4 (Lacrimal))     Nucleotide -   Genbank accession no. NM_007244 -   Genbank version no. NM_007244.2 GI:154448885 -   Genbank record update date: Jun. 28, 2012 12:39 PM     Polypeptide -   Genbank accession no. NP_009175 -   Genbank version no. NP_009175.2 GI:154448886 -   Genbank record update date: Jun. 28, 2012 12:39 PM     Cross-References -   Dickinson D. P., et al Invest. Ophthalmol. Vis. Sci. 36 (10),     2020-2031 (1995)     Other Information -   Official Symbol: PRR4 -   Other Aliases: LPRP, PROL4 -   Other Designations: lacrimal proline-rich protein; nasopharyngeal     carcinoma-associated proline-rich protein 4; proline-rich     polypeptide 4; proline-rich protein 4     (61) GCC—GUCY2C (Guanylate Cyclase 2C (Heat Stable Enterotoxin     Receptor)     Nucleotide -   Genbank accession no. NM_004963 -   Genbank version no. NM_004963.3 GI:222080082 -   Genbank record update date: Sep. 2, 2012 01:50 PM     Polypeptide -   Genbank accession no. NP_004954 -   Genbank version no. NP_004954.2 GI:222080083 -   Genbank record update date: Sep. 2, 2012 01:50 PM     Cross-References -   De Sauvage F. J., et al J. Biol. Chem. 266 (27), 17912-17918 (1991);     Singh S., et al Biochem. Biophys. Res. Commun. 179 (3), 1455-1463     (1991)     Other Information -   Official Symbol: GUCY2C -   Other Aliases: DIAR6, GUC2C, MUCIL, STAR -   Other Designations: GC-C; STA receptor; guanylyl cyclase C; hSTAR;     heat-stable enterotoxin receptor; intestinal guanylate cyclase     (62) Liv-1—SLC39A6 (Solute Carrier Family 39 (Zinc Transporter),     Member 6)     Nucleotide -   Genbank accession no. U41060 -   Genbank version no. U41060.2 GI:12711792 -   Genbank record update date: Nov. 30, 2009 04:35 PM     Polypeptide -   Genbank accession no. AAA96258 -   Genbank version no. AAA96258.2 GI:12711793 -   Genbank record update date: Nov. 30, 2009 04:35 PM     Cross-References -   Taylor K M., et al Biochim Biophys Acta. 2003 Apr. 1;     1611(1-2):16-30     Other Information -   Official Symbol: SLC39A6 -   Other Aliases: LIV-1 -   Other Designations: LIV-1 protein, estrogen regulated; ZIP-6;     estrogen-regulated protein LIV-1; solute carrier family 39 (metal     ion transporter), member 6; solute carrier family 39 member 6; zinc     transporter ZIP6; zrt- and Irt-like protein 6     (63) 5T4, Trophoblast Glycoprotein, TPBG—TPBG (Trophoblast     Glycoprotein)     Nucleotide -   Genbank accession no. AJ012159 -   Genbank version no. AJ012159.1 GI:3805946 -   Genbank record update date: Feb. 1, 2011 10:27 AM     Polypeptide -   Genbank accession no. CAA09930 -   Genbank version no. CAA09930.1 GI:3805947 -   Genbank record update date: Feb. 1, 2011 10:27 AM     Cross-References -   King K. W., et al Biochim. Biophys. Acta 1445 (3), 257-270 (1999)     Other Information     -   Official Symbol: TPBG     -   Other Aliases: 5T4, 5T4AG, M6P1     -   Other Designations: 5T4 oncofetal antigen; 5T4 oncofetal         trophoblast glycoprotein; 5T4 oncotrophoblast glycoprotein         (64) CD56—NCMA1 (Neural Cell Adhesion Molecule 1)         Nucleotide -   Genbank accession no. NM_000615 -   Genbank version no. NM_000615.6 GI:336285433 -   Genbank record update date: Sep. 23, 2012 02:32 PM     Polypeptide -   Genbank accession no. NP_000606 -   Genbank version no. NP_000606.3 GI:94420689 -   Genbank record update date: Sep. 23, 2012 02:32 PM     Cross-References -   Dickson, G., et al, Cell 50 (7), 1119-1130 (1987)     Other Information -   Official Symbol: NCAM1 -   Other Aliases: CD56, MSK39, NCAM -   Other Designations: antigen recognized by monoclonal antibody     5.1H11; neural cell adhesion molecule, NCAM     Antibodies -   Immunogen: HuN901 (Smith S V., et al Curr Opin Mol Ther. 2005 Aug.;     7(4):394-401)     -   For example, see humanized from murine N901 antibody. See FIGS.         1b and 1e of Roguska, M. A., et al. Proc Natl Acad Sci USA         February 1994; 91:969-973.         (65) CanAg (Tumor Associated Antigen CA242)         Cross-References -   Haglund C., et al Br J Cancer 60:845-851, 1989; Baeckstrom D., et al     J Biol Chem 266:21537-21547, 1991     Antibodies -   huC242 (Tolcher A W et al., J Clin Oncol. 2003 Jan. 15;     21(2):211-22; Immunogen)     -   For example, see US20080138898A1 SEQ ID NO: 1 and 2         (66) FOLR1 (Folate Receptor 1)         Nucleotide -   Genbank accession no. J05013 -   Genbank version no. J05013.1 GI:182417 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Polypeptide -   Genbank accession no. AAA35823 -   Genbank version no. AAA35823.1 GI:182418 -   Genbank record update date: Jun. 23, 2010 08:47 AM     Cross-References -   Elwood P. C., et al J. Biol. Chem. 264 (25), 14893-14901 (1989)     Other Information -   Official Symbol: FOLR1 -   Other Aliases: FBP, FOLR -   Other Designations: FR-alpha; KB cells FBP; adult folate-binding     protein; folate binding protein; folate receptor alpha; folate     receptor, adult; ovarian tumor-associated antigen MOv18     Antibodies -   M9346A—Whiteman K R., et al Cancer Res Apr. 15, 2012; 72(8     Supplement): 4628 (Immunogen)     (67) GPNMB (Glycoprotein (Transmembrane) nmb)     Nucleotide -   Genbank accession no. X76534 -   Genbank version no. X76534.1 GI:666042 -   Genbank record update date: Feb. 2, 2011 10:10 AM     Polypeptide -   Genbank accession no. CAA54044 -   Genbank version no. CAA54044.1 GI:666043 -   Genbank record update date: Feb. 2, 2011 10:10 AM     Cross-References -   Weterman M. A., et al Int. J. Cancer 60 (1), 73-81 (1995)     Other Information -   Official Symbol: GPNMB -   Other Aliases: UNQ1725/PRO9925, HGFIN, NMB -   Other Designations: glycoprotein NMB; glycoprotein nmb-like protein;     osteoactivin; transmembrane glycoprotein HGFIN; transmembrane     glycoprotein NMB     Antibodies -   Celldex Therapeutics: CR011 (Tse K F., et al Clin Cancer Res. 2006     Feb. 15; 12(4):1373-82)     -   For example, see EP1827492B1 SEQ ID NO: 22, 24, 26, 31, 33 and         35         (68) TIM-1—HAVCR1 (Hepatitis A Virus Cellular Receptor 1)         Nucleotide -   Genbank accession no. AF043724 -   Genbank version no. AF043724.1 GI:2827453 -   Genbank record update date: Mar. 10, 2010 06:24 PM     Polypeptide -   Genbank accession no. AAC39862 -   Genbank version no. AAC39862.1 GI:2827454 -   Genbank record update date: Mar. 10, 2010 06:24 PM     Cross-References -   Feigelstock D., et al J. Virol. 72 (8), 6621-6628 (1998)     Other Information -   Official Symbol: HAVCR1 -   Other Aliases: HAVCR, HAVCR-1, KIM-1, KIM1, TIM, TIM-1, TIM1,     TIMD-1, TIMD1 -   Other Designations: T cell immunoglobin domain and mucin domain     protein 1; T-cell membrane protein 1; kidney injury molecule 1     (69) RG-1/Prostate Tumor Target Mindin—Mindin/RG-1     Cross-References -   Parry R., et al Cancer Res. 2005 Sep. 15; 65(18):8397-405     (70) B7-H4—VTCN1 (V-Set Domain Containing T Cell Activation     Inhibitor 1     Nucleotide -   Genbank accession no. BX648021 -   Genbank version no. BX648021.1 GI:34367180 -   Genbank record update date: Feb. 2, 2011 08:40 AM     Cross-References -   Sica G L., et al Immunity. 2003 Jun.; 18(6):849-61     Other Information -   Official Symbol: VTCN1 -   Other Aliases: RP11-229A19.4, B7-H4, B7H4, B7S1, B7X, B7h.5,     PRO1291, VCTN1 -   Other Designations: B7 family member, H4; B7 superfamily member 1; T     cell costimulatory molecule B7x; T-cell costimulatory molecule B7x;     V-set domain-containing T-cell activation inhibitor 1; immune     costimulatory protein B7-H4     (71) PTK7 (PTK7 Protein Tyrosine Kinase 7)     Nucleotide -   Genbank accession no. AF447176 -   Genbank version no. AF447176.1 GI:17432420 -   Genbank record update date: Nov. 28, 2008 01:51 PM     Polypeptide -   Genbank accession no. AAL39062 -   Genbank version no. AAL39062.1 GI:17432421 -   Genbank record update date: Nov. 28, 2008 01:51 PM     Cross-References -   Park S. K., et al J. Biochem. 119 (2), 235-239 (1996)     Other Information -   Official Symbol: PTK7 -   Other Aliases: CCK-4, CCK4 -   Other Designations: colon carcinoma kinase 4; inactive     tyrosine-protein kinase 7; pseudo tyrosine kinase receptor 7;     tyrosine-protein kinase-like 7     (72) CD37 (CD37 Molecule)     Nucleotide -   Genbank accession no. NM_001040031 -   Genbank version no. NM_001040031.1 GI:91807109 -   Genbank record update date: Jul. 29, 2012 02:08 PM     Polypeptide -   Genbank accession no. NP_001035120 -   Genbank version no. NP_001035120.1 GI:91807110 -   Genbank record update date: Jul. 29, 2012 02:08 PM     Cross-References -   Schwartz-Albiez R., et al J. Immunol. 140 (3), 905-914 (1988)     Other Information -   Official Symbol: CD37 -   Other Aliases: GP52-40, TSPAN26 -   Other Designations: CD37 antigen; cell differentiation antigen 37;     leukocyte antigen CD37; leukocyte surface antigen CD37;     tetraspanin-26; tspan-26     Antibodies -   Boehringer Ingelheim: mAb 37.1 (Heider K H., et al Blood. 2011 Oct.     13; 118(15):4159-68) -   Trubion: CD37-SMIP (G28-1 scFv-lg) ((Zhao X., et al Blood. 2007;     110: 2569-2577)     -   For example, see US20110171208A1 SEQ ID NO: 253 -   Immunogen: K7153A (Deckert J., et al Cancer Res Apr. 15, 2012; 72(8     Supplement): 4625)     (73) CD138—SDC1 (Syndecan 1)     Nucleotide -   Genbank accession no. AJ551176 -   Genbank version no. AJ551176.1 GI:29243141 -   Genbank record update date: Feb. 1, 2011 12:09 PM     Polypeptide -   Genbank accession no. CAD80245 -   Genbank version no. CAD80245.1 GI:29243142 -   Genbank record update date: Feb. 1, 2011 12:09 PM     Cross-References -   O'Connell F P., et al Am J Clin Pathol. 2004 February; 121(2):254-63     Other Information -   Official Symbol: SDC1 -   Other Aliases: CD138, SDC, SYND1, syndecan -   Other Designations: CD138 antigen; heparan sulfate proteoglycan     fibroblast growth factor receptor; syndecan proteoglycan 1;     syndecan-1     Antibodies -   Biotest: chimerized MAb (nBT062)—(Jagannath S., et al Poster ASH     #3060, 2010; WIPO Patent Application WO/2010/128087)     -   For example, see US20090232810 SEQ ID NO: 1 and 2 Immunogen:         B-B4 (Tassone P., et al Blood 104_3688-3696)     -   For example, see US20090175863A1 SEQ ID NO: 1 and 2         (74) CD74 (CD74 Molecule, Major Histocompatibility Complex,         Class II Invariant Chain)         Nucleotide -   Genbank accession no. NM_004355 -   Genbank version no. NM_004355.1 GI:343403784 -   Genbank record update date: Sep. 23, 2012 02:30 PM     Polypeptide -   Genbank accession no. NP_004346 -   Genbank version no. NP_004346.1 GI:10835071 -   Genbank record update date: Sep. 23, 2012 02:30 PM     Cross-References -   Kudo, J., et al Nucleic Acids Res. 13 (24), 8827-8841 (1985)     Other Information -   Official Symbol: CD74 -   Other Aliases: DHLAG, HLADG, II, Ia-GAMMA -   Other Designations: CD74 antigen (invariant polypeptide of major     histocompatibility complex, class II antigen-associated); HLA class     II histocompatibility antigen gamma chain; HLA-DR     antigens-associated invariant chain; HLA-DR-gamma; Ia-associated     invariant chain; MHC HLA-DR gamma chain; gamma chain of class II     antigens; p33     Antibodies -   Immunomedics: hLL1 (Milatuzumab,)—Berkova Z., et al Expert Opin     Investig Drugs. 2010 Jan.; 19(1):141-9)     -   For example, see US20040115193 SEQ ID NOs: 19, 20, 21, 22, 23         and 24 -   Genmab: HuMax-CD74 (see website)     (75) Claudins—CLs (Claudins)     Cross-References -   Offner S., et al Cancer Immunol Immunother. 2005 May; 54(5):431-45,     Suzuki H., et al Ann NY Acad Sci. 2012 July; 1258:65-70) -   In humans, 24 members of the family have been described—see     literature reference.     (76) EGFR (Epidermal Growth Factor Receptor)     Nucleotide -   Genbank accession no. NM_005228 -   Genbank version no. NM_005228.3 GI:41927737 -   Genbank record update date: Sep. 30, 2012 01:47 PM     Polypeptide -   Genbank accession no. NP_005219 -   Genbank version no. NP_005219.2 GI:29725609 -   Genbank record update date: Sep. 30, 2012 01:47 PM     Cross-References -   Dhomen N S., et al Crit Rev Oncog. 2012; 17(1):31-50     Other Information -   Official Symbol: EGFR -   Other Aliases: ERBB, ERBB1, HER1, PIG61, mENA -   Other Designations: avian erythroblastic leukemia viral (v-erb-b)     oncogene homolog; cell growth inhibiting protein 40; cell     proliferation-inducing protein 61; proto-oncogene c-ErbB-1; receptor     tyrosine-protein kinase erbB-1     Antibodies -   BMS: Cetuximab (Erbitux)—Broadbridge V T., et al Expert Rev     Anticancer Ther. 2012 May; 12(5):555-65.     -   For example, see U.S. Pat. No. 6,217,866—ATTC deposit No. 9764. -   Amgen: Panitumumab (Vectibix)—Argiles G., et al Future Oncol. 2012     Apr.; 8(4):373-89     -   For example, see U.S. Pat. No. 6,235,883 SEQ ID NOs: 23-38. -   Genmab: Zalutumumab—Rivera F., et al Expert Opin Biol Ther. 2009     May; 9(5):667-74. -   YM Biosciences: Nimotuzumab—Ramakrishnan M S., et al MAbs. 2009     Jan.-Feb.; 1(1):41-8.     -   For example, see U.S. Pat. No. 5,891,996 SEQ ID NOs: 27-34.         (77) Her3 (ErbB3)—ERBB3 (v-Erb-b2 Erythroblastic Leukemia Viral         Oncogene Homolog 3 (Avian))         Nucleotide -   Genbank accession no. M34309 -   Genbank version no. M34309.1 GI:183990 -   Genbank record update date: Jun. 23, 2010 08:47 PM     Polypeptide -   Genbank accession no. AAA35979 -   Genbank version no. AAA35979.1 GI:306841 -   Genbank record update date: Jun. 23, 2010 08:47 PM     Cross-References -   Plowman, G. D., et al., Proc. Natl. Acad. Sci. U.S.A. 87 (13),     4905-4909 (1990)     Other Information -   Official Symbol: ERBB3 -   Other Aliases: ErbB-3, HER3, LCCS2, MDA-BF-1, c-erbB-3, c-erbB3,     erbB3-S, p180-ErbB3, p45-sErbB3, p85-sErbB3 -   Other Designations: proto-oncogene-like protein c-ErbB-3; receptor     tyrosine-protein kinase erbB-3; tyrosine kinase-type cell surface     receptor HER3     Antibodies -   Merimack Pharma: MM-121 (Schoeberl B., et al Cancer Res. 2010 Mar.     15; 70(6):2485-2494)     -   For example, see US2011028129 SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7         and 8.         (78) RON—MST1R (Macrophage Stimulating 1 Receptor (c-Met-Related         Tyrosine Kinase))         Nucleotide -   Genbank accession no. X70040 -   Genbank version no. X70040.1 GI:36109 -   Genbank record update date: Feb. 2, 2011 10:17 PM     Polypeptide -   Genbank accession no. CCA49634 -   Genbank version no. CCA49634.1 GI:36110 -   Genbank record update date: Feb. 2, 2011 10:17 PM     Cross-References -   Ronsin C., et al Oncogene 8 (5), 1195-1202 (1993)     Other Information -   Official Symbol: MST1R -   Other Aliases: CD136, CDw136, PTK8, RON -   Other Designations: MSP receptor; MST1R variant RON30; MST1R variant     RON62; PTK8 protein tyrosine kinase 8; RON variant E2E3;     c-met-related tyrosine kinase; macrophage-stimulating protein     receptor; p185-Ron; soluble RON variant 1; soluble RON variant 2;     soluble RON variant 3; soluble RONvariant 4     (79) EPHA2 (EPH Receptor A2)     Nucleotide -   Genbank accession no. BC037166 -   Genbank version no. BC037166.2 GI:33879863 -   Genbank record update date: Mar. 6, 2012 01:59 PM     Polypeptide -   Genbank accession no. AAH37166 -   Genbank version no. AAH37166.1 GI:22713539 -   Genbank record update date: Mar. 6, 2012 01:59 PM     Cross-References -   Strausberg R. L., et al Proc. Natl. Acad. Sci. U.S.A. 99 (26),     16899-16903 (2002)     Other Information -   Official Symbol: EPHA2 -   Other Aliases: ARCC2, CTPA, CTPP1, ECK -   Other Designations: ephrin type-A receptor 2; epithelial cell     receptor protein tyrosine kinase; soluble EPHA2 variant 1;     tyrosine-protein kinase receptor ECK     Antibodies -   Medimmune: 1C1 (Lee J W., et al Clin Cancer Res. 2010 May 1;     16(9):2562-2570)     -   For example, see US20090304721A1 FIGS. 7 and 8.         (80) CD20—MS4A1 (Membrane-Spanning 4-Domains, Subfamily A,         Member 1)         Nucleotide -   Genbank accession no. M27394 -   Genbank version no. M27394.1 GI:179307 -   Genbank record update date: Nov. 30, 2009 11:16 AM     Polypeptide -   Genbank accession no. AAA35581 -   Genbank version no. AAA35581.1 GI:179308 -   Genbank record update date: Nov. 30, 2009 11:16 AM     Cross-References -   Tedder T. F., et al Proc. Natl. Acad. Sci. U.S.A. 85 (1), 208-212     (1988)     Other Information -   Official Symbol: MS4A1 -   Other Aliases: B1, Bp35, CD20, CVID5, LEU-16, MS4A2, S7 -   Other Designations: B-lymphocyte antigen CD20; B-lymphocyte     cell-surface antigen B1; CD20 antigen; CD20 receptor; leukocyte     surface antigen Leu-16     Antibodies -   Genentech/Roche: Rituximab—Abdulla N E., et al BioDrugs. 2012 Apr.     1; 26(2):71-82.     -   For example, see U.S. Pat. No. 5,736,137, ATCC deposit No.         HB-69119. -   GSK/Genmab: Ofatumumab—Nightingale G., et al Ann Pharmacother. 2011     October; 45(10):1248-55.     -   For example, see US20090169550A1 SEQ ID NOs: 2, 4 and 5. -   Immunomedics: Veltuzumab—Goldenberg D M., et al Leuk Lymphoma. 2010     May; 51(5):747-55.     -   For example, see U.S. Pat. No. 7,919,273B2 SEQ ID NOs: 1, 2, 3,         4, 5 and 6.         (81) Tenascin C—TNC (Tenascin C)         Nucleotide -   Genbank accession no. NM_002160 -   Genbank version no. NM_002160.3 GI:340745336 -   Genbank record update date: Sep. 23, 2012 02:33 PM     Polypeptide -   Genbank accession no. NP_002151 -   Genbank version no. NP_002151.2 GI:153946395 -   Genbank record update date: Sep. 23, 2012 02:33 PM     Cross-References -   Nies D. E., et al J. Biol. Chem. 266 (5), 2818-2823 (1991); Siri A.,     et al Nucleic Acids Res. 19 (3), 525-531 (1991)     Other Information -   Official Symbol: TNC -   Other Aliases: 150-225, GMEM, GP, HXB, JI, TN, TN-C -   Other Designations: GP 150-225; cytotactin;     glioma-associated-extracellular matrix antigen; hexabrachion     (tenascin); myotendinous antigen; neuronectin; tenascin; tenascin-C     isoform 14/AD1/16     Antibodies -   Philogen: G11 (von Lukowicz T., et al J Nucl Med. 2007 April;     48(4):582-7) and F16 (Pedretti M., et al Lung Cancer. 2009 April;     64(1):28-33)     -   For example, see U.S. Pat. No. 7,968,685 SEQ ID NOs: 29, 35, 45         and 47.         (82) FAP (Fibroblast Activation Protein, Alpha)         Nucleotide -   Genbank accession no. U09278 -   Genbank version no. U09278.1 GI:1888315 -   Genbank record update date: Jun. 23, 2010 09:22 AM     Polypeptide -   Genbank accession no. AAB49652 -   Genbank version no. AAB49652.1 GI:1888316 -   Genbank record update date: Jun. 23, 2010 09:22 AM     Cross-References -   Scanlan, M. J., et al Proc. Natl. Acad. Sci. U.S.A. 91 (12),     5657-5661 (1994)     Other Information -   Official Symbol: FAP -   Other Aliases: DPPIV, FAPA -   Other Designations: 170 kDa melanoma membrane-bound gelatinase;     integral membrane serine protease; seprase     (83) DKK-1 (Dickkopf 1 Homolog (Xenopus laevis)     Nucleotide -   Genbank accession no. NM_012242 -   Genbank version no. NM_012242.2 GI:61676924 -   Genbank record update date: Sep. 30, 2012 01:48 PM     Polypeptide -   Genbank accession no. NP_036374 -   Genbank version no. NP_036374.1 GI:7110719 -   Genbank record update date: Sep. 30, 2012 01:48 PM     Cross-References -   Fedi P. et al J. Biol. Chem. 274 (27), 19465-19472 (1999)     Other Information -   Official Symbol: DKK1 -   Other Aliases: UNQ492/PRO1008, DKK-1, SK -   Other Designations: dickkopf related protein-i; dickkopf-1 like;     dickkopf-like protein 1; dickkopf-related protein 1; hDkk-1     Antibodies -   Novartis: BHQ880 (Fulciniti M., et al Blood. 2009 Jul. 9;     114(2):371-379)     -   For example, see US20120052070A1 SEQ ID NOs: 100 and 108. (84)         CD52 (CD52 molecule)         Nucleotide -   Genbank accession no. NM_001803 -   Genbank version no. NM_001803.2 GI:68342029 -   Genbank record update date: Sep. 30, 2012 01:48 PM     Polypeptide -   Genbank accession no. NP_001794 -   Genbank version no. NP_001794.2 GI:68342030 -   Genbank record update date: Sep. 30, 2012 01:48 PM     Cross-References -   Xia M. Q., et al Eur. J. Immunol. 21 (7), 1677-1684 (1991)     Other Information -   Official Symbol: CD52 -   Other Aliases: CDW52 -   Other Designations: CAMPATH-1 antigen; CD52 antigen (CAMPATH-1     antigen); CDW52 antigen (CAMPATH-1 antigen); cambridge pathology 1     antigen; epididymal secretory protein E5; he5; human     epididymis-specific protein 5     Antibodies -   Alemtuzumab (Campath)—Skoetz N., et al Cochrane Database Syst Rev.     2012 Feb. 15; 2:CD008078.     -   For example, see Drugbank Acc. No. DB00087 (BIOD00109, BTD00109)         (85) CS1—SLAMF7 (SLAM Family Member 7)         Nucleotide -   Genbank accession no. NM_021181 -   Genbank version no. NM_021181.3 GI:1993571 -   Genbank record update date: Jun. 29, 2012 11:24 AM     Polypeptide -   Genbank accession no. NP_067004 -   Genbank version no. NP_067004.3 GI:19923572 -   Genbank record update date: Jun. 29, 2012 11:24 AM     Cross-References -   Boles K. S., et al Immunogenetics 52 (3-4), 302-307 (2001)     Other Information -   Official Symbol: SLAMF7 -   Other Aliases: UNQ576/PRO1138, 19A, CD319, CRACC, CS1 -   Other Designations: 19A24 protein; CD2 subset 1; CD2-like receptor     activating cytotoxic cells; CD2-like receptor-activating cytotoxic     cells; membrane protein FOAP-12; novel LY9 (lymphocyte antigen 9)     like protein; protein 19A     Antibodies -   BMS: elotuzumab/HuLuc63 (Benson D M., et al J Clin Oncol. 2012 Jun.     1; 30(16):2013-2015)     -   For example, see US20110206701 SEQ ID NOs: 9, 10, 11, 12, 13,         14, 15 and 16.         (86) Endoglin—ENG (Endoglin)         Nucleotide -   Genbank accession no. AF035753 -   Genbank version no. AF035753.1 GI:3452260 -   Genbank record update date: Mar. 10, 2010 06:36 PM     Polypeptide -   Genbank accession no. AAC32802 -   Genbank version no. AAC32802.1 GI:3452261 -   Genbank record update date: Mar. 10, 2010 06:36 PM     Cross-References -   Rius C., et al Blood 92 (12), 4677-4690 (1998) -   Official Symbol: ENG     Other Information -   Other Aliases: RP11-228B15.2, CD105, END, HHT1, ORW, ORW1 -   Other Designations: CD105 antigen     (87) Annexin A1—ANXA1 (Annexin A1)     Nucleotide -   Genbank accession no. X05908 -   Genbank version no. X05908.1 GI:34387 -   Genbank record update date: Feb. 2, 2011 10:02 AM     Polypeptide -   Genbank accession no. CCA29338 -   Genbank version no. CCA29338.1 GI:34388 -   Genbank record update date: Feb. 2, 2011 10:02 AM     Cross-References -   Wallner B. P., et al Nature 320 (6057), 77-81 (1986)     Other Information -   Official Symbol: ANXA1 -   Other Aliases: RP11-71A24.1, ANX1, LPC1 -   Other Designations: annexin I (lipocortin I); annexin-1; calpactin     II; calpactin-2; chromobindin-9; lipocortin I; p35; phospholipase A2     inhibitory protein     (88) V-CAM (CD106)—VCAM1 (Vascular Cell Adhesion Molecule 1)     Nucleotide -   Genbank accession no. M60335 -   Genbank version no. M60335.1 GI:340193 -   Genbank record update date: Jun. 23, 2010 08:56 AM     Polypeptide -   Genbank accession no. AAA61269 -   Genbank version no. AAA61269.1 GI:340194 -   Genbank record update date: Jun. 23, 2010 08:56 AM     Cross-References -   Hession C., et al J. Biol. Chem. 266 (11), 6682-6685 (1991)     Other Information -   Official Symbol VCAM1 -   Other Aliases: CD106, INCAM-100 -   Other Designations: CD106 antigen; vascular cell adhesion protein 1

Antibody Sequences Anti-Integrin avβ6 RHAB6.2 QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTE YAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTAVPNLRGDLQVLAQKVAG PYPFDYWGQGTLVTVSS RHCB6.2 QVQLVQSGAEVKKPGASVKVSCKASGYTFIDSYMHWVRQAPGQRLEWMGWIDPENGDTE YAPKFQGRVTITTDTSASTAYMELSSLRSEDTAVYYCARGTPTAVPNLRGDLQVLAQKVAG PYPFDYWGQGTLVTVSS RHF QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDT EYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTGPYYFDYWGQGTLVTV SS RHFB6 QVQLVQSGAEVKKPGASVKVSCKASGFNFIDSYMHWVRQAPGQRLEWMGWIDPENGDT EYAPKFQGRVTFTTDTSASTAYMELSSLRSEDTAVYYCNEGTPTAVPNLRGDLQVLAQKVA GPYYFDYWGQGTLVTVSS RHAY100bP QVQLVQSGSELKKPGASVKISCKASGFAFTDSYMHWVRQAPGQGLEWMGWIDPENGDTE YAPKFQGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCTRGTPTGPYPFDYWGQGTLVTVSS RKF ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKFL36L50 ENVLTQSPGTLSLSPGERATLSCSASSSVSYMHWLQQKPGQAPRLLIYLTSNLASGIPDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK RKC EIVLTQSPGTLSLSPGERATLSCSASSSVSYMHWFQQKPGQAPRLLIYSTSNLASGIPDRFS GSGSGTDFTLTISRLEPEDFAVYYCQQRSSYPLTFGGGTKVEIK Anti-CD33 CD33 Hum195 VH QVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYNMHWVRQAPGQGLEWIGYIYPYNGGTG YNQKFKSKATITADESTNTAYMELSSLRSEDTAVYYCARGRPAMDYWGQGTLVTVSS CD33 Hum195 VK DIQMTQSPSSLSASVGDRVTITCRASESVDNYGISFMNWFQQKPGKAPKLLIYAASNQGSG VPSRFSGSGSGTDFTLTISSLQPDDFATYYCQQSKEVPWTFGQGTKVEIK Anti-CD19 CD19 B4 resurfaced VH QVQLVQPGAEVVKPGASVKLSCKTSGYTFTSNWMHWVKQRPGQGLEWIGEIDPSDSYTN YNQNFKGKAKLTVDKSTSTAYMEVSSLRSDDTAVYYCARGSNPYYYAMDYWGQGTSVTV SS CD19 B4 resurfaced VK EIVLTQSPAIMSASPGERVTMTCSASSGVNYMHWYQQKPGTSPRRWIYDTSKLASGVPAR FSGSGSGTSYSLTISSMEPEDAATYYCHQRGSYTFGGGTKLEIK Anti-Her2 Herceptin VH chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVS S Herceptin VL chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR FSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK Anti-CD25 Simulect VK (also known as Basiliximab) QIVSTQSPAIMSASPGEKVTMTCSASSSRSYMQWYQQKPGTSPKRWIYDTSKLASGVPAR FSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYTFGGGTKLEIK Simulect VH QLQQSGTVLARPGASVKMSCKASGYSFTRYWMHWIKQRPGQGLEWIGAIYPGNSDTSYN QKFEGKAKLTAVTSASTAYMELSSLTHEDSAVYYCSRDYGYYFDFWGQGTTLTVSS Anti-PSMA Deimmunised VH ′1 EVQLVQSGPEVKKPGATVKISCKTSGYTFTEYTIHWVKQAPGKGLEWIGNINPNNGGTTYN QKFEDKATLTVDKSTDTAYMELSSLRSEDTAVYYCAAGWNFDYWGQGTLLTVSS Deimmunised VK ′1 DIQMTQSPSSLSTSVGDRVTLTCKASQDVGTAVDWYQQKPGPSPKLLIYWASTRHTGIPSR FSGSGSGTDFTLTISSLQPEDFADYYCQQYNSYPLTFGPGTKVDIK Deimmunised VH1 ′5 EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNF ATHYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTGVYYCTRRWNNFWGQGTTVTVSS Deimmunised VH2 ′5 EVKLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFA THYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS Deimmunised VH3 ′5 EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFA THYAESVKGRVTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS Deimmunised VH4 ′5 EVQLVESGGGLVQPGGSLKLSCVASGFTFSNYWMNWVRQAPGKGLEWVAEIRSQSNNFA THYAESVKGRFTISRDDSKSIVYLQMNNLRAEDTAVYYCTRRWNNFWGQGTTVTVSS Deimmunised VK1 ′5 NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPD RFTGSGSATDFTLTISSLQTEDLADYYCGQSYTFPYTFGQGTKLEMK Deimmunised VK2 ′5 NIVMTQFPSSMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPD RFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK3 ′5 NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPD RFSGSGSGTDFTLTISSLQAEDLADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK4 ′5 NIQMTQFPSAMSASVGDRVTITCKASENVGTYVSWYQQKPDQSPKMLIYGASNRFTGVPD RFSGSGSGTDFTLTISSLQAEDEADYYCGQSYTFPYTFGQGTKLEIK Deimmunised VK DI ′5 NIVMTQFPKSMSASAGERMTLTCKASENVGTYVSWYQQKPTQSPKMLIYGASNRFTGVPD RFSGSGSGTDFILTISSVQAEDLVDYYCGQSYTFPYTFGGGTKLEMK Deimmunised VH DI ′5 EVKLEESGGGLVQPGGSMKISCVASGFTFSNYWMNWVRQSPEKGLEWVAEIRSQSNNFA THYAESVKGRVIISRDDSKSSVYLQMNSLRAEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHA ′5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFA THYAESVKGRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHB ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHC ′5 EVQLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHD ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVGEIRSQSNNFA THYAESVKGRVIISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHE ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRFTISRDDSKNTVYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHF ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RHG ′5 EVKLVESGGGLVQPGGSLKLSCAASGFTFSNYWMNWVRQASGKGLEWVAEIRSQSNNFA THYAESVKGRVIISRDDSKNTAYLQMNSLRTEDTAVYYCTRRWNNFWGQGTTVTVSS Humanised RKA ′5 DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSR FSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKB ′5 DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPSR FSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKC ′5 DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPS RFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKD ′5 DIQMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPS RFSGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKE ′5 NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKLLIYGASNRFTGVPDR FTGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKF ′5 NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPSR FSGSGSATDFILTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK Humanised RKG ′5 NIVMTQSPSSVSASVGDRVTITCKASENVGTYVSWYQQKPGTAPKMLIYGASNRFTGVPDR FTGSGSATDFTLTINNLQPEDFATYYCGQSYTFPYTFGQGTKVEIK

The parent antibody may also be a fusion protein comprising an albumin-binding peptide (ABP) sequence (Dennis et al. (2002) “Albumin Binding As A General Strategy For Improving The Pharmacokinetics Of Proteins” J Biol Chem. 277:35035-35043; WO 01/45746). Antibodies of the invention include fusion proteins with ABP sequences taught by: (i) Dennis et al (2002) J Biol Chem. 277:35035-35043 at Tables III and IV, page 35038; (ii) US 2004/0001827 at [0076]; and (iii) WO 01/45746 at pages 12-13, and all of which are incorporated herein by reference.

In one embodiment, the antibody has been raised to target specific the tumour related antigen α_(v)β₆.

The cell binding agent may be labelled, for example to aid detection or purification of the agent either prior to incorporation as a conjugate, or as part of the conjugate. The label may be a biotin label. In another embodiment, the cell binding agent may be labelled with a radioisotope.

Embodiments of the present invention include ConjA wherein the cell binding agent is selected from an antibody to any of the antigens discussed above.

Embodiments of the present invention include ConjB wherein the cell binding agent is selected from an antibody to any of the antigens discussed above.

Embodiments of the present invention include ConjA wherein the cell binding agent is selected from any of the antibodies discussed above.

Embodiments of the present invention include ConjB wherein the cell binding agent is selected from any of the antibodies discussed above.

The present invention may also relate to conjugates where the cell binding agent is selected from an antibody to any of the antigens discussed above and any of the antibodies discussed above linked to different drugs.

Drug Loading

The drug loading is the average number of PBD drugs per cell binding agent, e.g. antibody. Where the compounds of the invention are bound to cysteines, drug loading may range from 1 to 8 drugs (D) per cell binding agent, i.e. where 1, 2, 3, 4, 5, 6, 7, and 8 drug moieties are covalently attached to the cell binding agent. Compositions of conjugates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 8. Where the compounds of the invention are bound to lysines, drug loading may range from 1 to 80 drugs (D) per cell binding agent, although an upper limit of 40, 20, 10 or 8 may be preferred. Compositions of conjugates include collections of cell binding agents, e.g. antibodies, conjugated with a range of drugs, from 1 to 80, 1 to 40, 1 to 20, 1 to 10 or 1 to 8.

The average number of drugs per antibody in preparations of ADC from conjugation reactions may be characterized by conventional means such as UV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, and electrophoresis. The quantitative distribution of ADC in terms of p may also be determined. By ELISA, the averaged value of p in a particular preparation of ADC may be determined (Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852). However, the distribution of p (drug) values is not discernible by the antibody-antigen binding and detection limitation of ELISA. Also, ELISA assay for detection of antibody-drug conjugates does not determine where the drug moieties are attached to the antibody, such as the heavy chain or light chain fragments, or the particular amino acid residues. In some instances, separation, purification, and characterization of homogeneous ADC where p is a certain value from ADC with other drug loadings may be achieved by means such as reverse phase HPLC or electrophoresis. Such techniques are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number of attachment sites on the antibody. For example, an antibody may have only one or several cysteine thiol groups, or may have only one or several sufficiently reactive thiol groups through which a linker may be attached. Higher drug loading, e.g. p>5, may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates.

Typically, fewer than the theoretical maximum of drug moieties are conjugated to an antibody during a conjugation reaction. An antibody may contain, for example, many lysine residues that do not react with the drug-linker intermediate (D-L) or linker reagent. Only the most reactive lysine groups may react with an amine-reactive linker reagent. Also, only the most reactive cysteine thiol groups may react with a thiol-reactive linker reagent. Generally, antibodies do not contain many, if any, free and reactive cysteine thiol groups which may be linked to a drug moiety. Most cysteine thiol residues in the antibodies of the compounds exist as disulfide bridges and must be reduced with a reducing agent such as dithiothreitol (DTT) or TCEP, under partial or total reducing conditions. The loading (drug/antibody ratio) of an ADC may be controlled in several different manners, including: (i) limiting the molar excess of drug-linker intermediate (D-L) or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, and (iii) partial or limiting reductive conditions for cysteine thiol modification.

Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Each cysteine bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol. Reactive thiol groups may be introduced into the antibody (or fragment thereof) by engineering one, two, three, four, or more cysteine residues (e.g., preparing mutant antibodies comprising one or more non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541 teaches engineering antibodies by introduction of reactive cysteine amino acids.

Cysteine amino acids may be engineered at reactive sites in an antibody and which do not form intrachain or intermolecular disulfide linkages (Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al (2009) Blood 114(13):2721-2729; U.S. Pat. No. 7,521,541; U.S. Pat. No. 7,723,485; WO2009/052249). The engineered cysteine thiols may react with linker reagents or the drug-linker reagents of the present invention which have thiol-reactive, electrophilic groups such as maleimide or alpha-halo amides to form ADC with cysteine engineered antibodies and the PBD drug moieties. The location of the drug moiety can thus be designed, controlled, and known. The drug loading can be controlled since the engineered cysteine thiol groups typically react with thiol-reactive linker reagents or drug-linker reagents in high yield. Engineering an IgG antibody to introduce a cysteine amino acid by substitution at a single site on the heavy or light chain gives two new cysteines on the symmetrical antibody.

A drug loading near 2 can be achieved with near homogeneity of the conjugation product ADC.

Where more than one nucleophilic or electrophilic group of the antibody reacts with a drug-linker intermediate, or linker reagent followed by drug moiety reagent, then the resulting product is a mixture of ADC compounds with a distribution of drug moieties attached to an antibody, e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymeric reverse phase (PLRP) and hydrophobic interaction (HIC) may separate compounds in the mixture by drug loading value. Preparations of ADC with a single drug loading value (p) may be isolated, however, these single loading value ADCs may still be heterogeneous mixtures because the drug moieties may be attached, via the linker, at different sites on the antibody.

Thus the antibody-drug conjugate compositions of the invention include mixtures of antibody-drug conjugate compounds where the antibody has one or more PBD drug moieties and where the drug moieties may be attached to the antibody at various amino acid residues.

In one embodiment, the average number of dimer pyrrolobenzodiazepine groups per cell binding agent is in the range 1 to 20. In some embodiments the range is selected from 1 to 8, 2 to 8, 2 to 6, 2 to 4, and 4 to 8.

In some embodiments, there is one dimer pyrrolobenzodiazepine group per cell binding agent.

Preferred Compounds

Particularly preferred compounds of the first aspect of the present invention include:

Particularly preferred compounds of the second aspect of the present invention include:

Particularly preferred compounds of the third aspect of the present invention include:

Substituents

The phrase “optionally substituted” as used herein, pertains to a parent group which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein, pertains to a parent group which bears one or more substituents. The term “substituent” is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, or if appropriate, fused to, a parent group. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.

In a preferred embodiment, the substituents described herein (which include optional substituents) are limited to those groups that are not reactive to a cell binding agent. The link to the cell binding agent in the present case is formed from the bridge between the two PBD moieties through a linker group to the cell binding agent. Reactive functional groups located at other parts of the PBD structure may be capable of forming additional bonds to the cell binding agent (this may be referred to as crosslinking). These additional bonds may alter transport and biological activity of the conjugate. Therefore, in some embodiment, the additional substituents are limited to those lacking reactive functionality.

In one embodiment, the substituents are selected from the group consisting of R, OR, SR, NRR′, NO₂, halo, CO₂R, COR, CONH₂, CONHR, and CONRR′.

In one embodiment, the substituents are selected from the group consisting of R, OR, SR, NRR′, NO₂, CO₂R, COR, CONH₂, CONHR, and CONRR′.

In one embodiment, the substituents are selected from the group consisting of R, OR, SR, NRR′, NO₂, and halo.

In one embodiment, the substituents are selected from the group consisting of R, OR, SR, NRR′, and NO₂.

Any one of the embodiment mentioned above may be applied to any one of the substituents described herein. Alternatively, the substituents may be selected from one or more of the groups listed below.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 1 to 12 carbon atoms, which may be aliphatic or alicyclic, and which may be saturated or unsaturated (e.g. partially unsaturated, fully unsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl, alkynyl, cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to, methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl (C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limited to, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl (amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃), iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), and neo-pentyl (C₅).

An alkyl group may optionally be interrupted by one or more heteroatoms selected from O, N(H) and S. Such groups may be referred to as “heteroalkyl”.

C₂₋₁₂ Heteroalkyl: The term “C₂₋₁₂ heteroalkyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a carbon atom of a hydrocarbon compound having from 2 to 12 carbon atoms, and one or more heteroatoms selected from O, N(H) and S, preferably O and S.

Examples of heteroalkyl groups include, but are not limited to those comprising one or more ethylene glycol units of the type —(OCH₂CH₂)—. The terminal of a heteroalkyl group may be the primary form of a heteroatom, e.g. —OH, —SH or —NH₂. In a preferred embodiment, the terminal is —CH₃.

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to an alkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to, ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl, —CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄), pentenyl (C₅), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to an alkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to, ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertains to an alkyl group which is also a cyclyl group; that is, a monovalent moiety obtained by removing a hydrogen atom from an alicyclic ring atom of a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3 to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, those derived from:

-   -   saturated monocyclic hydrocarbon compounds:         cyclopropane (C₃), cyclobutane (C₄), cyclopentane (C₅),         cyclohexane (C₆), cycloheptane (C₇), methylcyclopropane (C₄),         dimethylcyclopropane (C₅), methylcyclobutane (C₅),         dimethylcyclobutane (C₆), methylcyclopentane (C₆),         dimethylcyclopentane (C₇) and methylcyclohexane (C₇);     -   unsaturated monocyclic hydrocarbon compounds:         cyclopropene (C₃), cyclobutene (C₄), cyclopentene (C₅),         cyclohexene (C₆), methylcyclopropene (C₄), dimethylcyclopropene         (C₅), methylcyclobutene (C₅), dimethylcyclobutene (C₆),         methylcyclopentene (C₆), dimethylcyclopentene (C₇) and         methylcyclohexene (C₇); and     -   saturated polycyclic hydrocarbon compounds:         norcarane (C₇), norpinane (C₇), norbornane (C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from a ring atom of a heterocyclic compound, which moiety has from 3 to 20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably, each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ring heteroatoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₃₋₇, C₅₋₆, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C₅₋₆heterocyclyl”, as used herein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limited to, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole) (C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrrole or 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆), dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇); O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole (dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆), pyran (C₆), oxepin (C₇); S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅), thiane (tetrahydrothiopyran) (C₆), thiepane (C₇); O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇); O₃: trioxane (C₆); N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline (C₅), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆); N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole (C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆), dihydrooxazine (C₆), oxazine (C₆); N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆); N₂O₁: oxadiazine (C₆); O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and, N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include those derived from saccharides, in cyclic form, for example, furanoses (C₅), such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse, and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose, mannopyranose, gulopyranose, idopyranose, galactopyranose, and talopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to a monovalent moiety obtained by removing a hydrogen atom from an aromatic ring atom of an aromatic compound, which moiety has from 3 to 20 ring atoms. Preferably, each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, etc.) denote the number of ring atoms, or range of number of ring atoms, whether carbon atoms or heteroatoms. For example, the term “C₅₋₆ aryl” as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, those derived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene (C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), and pyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one of which is an aromatic ring, include, but are not limited to, groups derived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉), isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀), acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene (C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in “heteroaryl groups”. Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅);

N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅), pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g., cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₆); and,

N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are not limited to:

C₉ (with 2 fused rings) derived from benzofuran (O₁), isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g., adenine, guanine), benzimidazole (N₂), indazole (N₂), benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (C₂), benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁), benzothiazole (N₁S₁), benzothiadiazole (N₂S); C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁), benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂), quinazoline (N₂), cinnoline (N₂), phthalazine (N₂), naphthyridine (N₂), pteridine (N₄); C₁₁ (with 2 fused rings) derived from benzodiazepine (N₂); C₁₃ (with 3 fused rings) derived from carbazole (N₁), dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂), perimidine (N₂), pyridoindole (N₂); and, C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene (O₁), thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁), phenazine (N₂), phenoxazine (N₁O₁), phenothiazine (N₁S₁), thianthrene (S₂), phenanthridine (N₁), phenanthroline (N₂), phenazine (N₂).

The above groups, whether alone or part of another substituent, may themselves optionally be substituted with one or more groups selected from themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkyl group (also referred to as a C₁₋₇ alkoxy group, discussed below), a C₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxy group), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxy group), preferably a C₁₋₇alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkyl group. Examples of C₁₋₇ alkoxy groups include, but are not limited to, —OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr) (isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu) (isobutoxy), and —O(tBu) (tert-butoxy).

Acetal: —CH(OR¹)(OR²), wherein R¹ and R² are independently acetal substituents, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, or, in the case of a “cyclic” acetal group, R¹ and R², taken together with the two oxygen atoms to which they are attached, and the carbon atoms to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Examples of acetal groups include, but are not limited to, —CH(OMe)₂, —CH(OEt)₂, and —CH(OMe)(OEt).

Hemiacetal: —CH(OH)(OR¹), wherein R¹ is a hemiacetal substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include, but are not limited to, —CH(OH)(OMe) and —CH(OH)(OEt).

Ketal: —CR(OR¹)(OR²), where R¹ and R² are as defined for acetals, and R is a ketal substituent other than hydrogen, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples ketal groups include, but are not limited to, —C(Me)(OMe)₂, —C(Me)(OEt)₂, —C(Me)(OMe)(OEt), —C(Et)(OMe)₂, —C(Et)(OEt)₂, and —C(Et)(OMe)(OEt).

Hemiketal: —CR(OH)(OR¹), where R¹ is as defined for hemiacetals, and R is a hemiketal substituent other than hydrogen, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of hemiacetal groups include, but are not limited to, —C(Me)(OH)(OMe), —C(Et)(OH)(OMe), —C(Me)(OH)(OEt), and —C(Et)(OH)(OEt).

Oxo (keto, -one): ═O.

Thione (thioketone): ═S.

Imino (imine): ═NR, wherein R is an imino substituent, for example, hydrogen, C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples of ester groups include, but are not limited to, ═NH, ═NMe, ═NEt, and ═NPh.

Formyl (carbaldehyde, carboxaldehyde): —C(═O)H.

Acyl (keto): —C(═O)R, wherein R is an acyl substituent, for example, a C₁₋₇ alkyl group (also referred to as C₁₋₇ alkylacyl or C₁₋₇ alkanoyl), a C₃₋₂₀ heterocyclyl group (also referred to as C₃₋₂₀ heterocyclylacyl), or a C₅₋₂₀ aryl group (also referred to as C₅₋₂₀ arylacyl), preferably a C₁₋₇ alkyl group. Examples of acyl groups include, but are not limited to, —C(═O)CH₃ (acetyl), —C(═O)CH₂CH₃ (propionyl), —C(═O)C(CH₃)₃ (t-butyryl), and —C(═O)Ph (benzoyl, phenone).

Carboxy (carboxylic acid): —C(═O)OH.

Thiocarboxy (thiocarboxylic acid): —C(═S)SH.

Thiolocarboxy (thiolocarboxylic acid): —C(═O)SH.

Thionocarboxy (thionocarboxylic acid): —C(═S)OH.

Imidic acid: —C(═NH)OH.

Hydroxamic acid: —C(═NOH)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR, wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of ester groups include, but are not limited to, —C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Acyloxy (reverse ester): —OC(═O)R, wherein R is an acyloxy substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of acyloxy groups include, but are not limited to, —OC(═O)CH₃ (acetoxy), —OC(═O)CH₂CH₃, —OC(═O)C(CH₃)₃, —OC(═O)Ph, and —OC(═O)CH₂Ph.

Oxycarboyloxy: —OC(═O)OR, wherein R is an ester substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of ester groups include, but are not limited to, —OC(═O)OCH₃, —OC(═O)OCH₂CH₃, —OC(═O)OC(CH₃)₃, and —OC(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents, for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇ alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group, or, in the case of a “cyclic” amino group, R¹ and R², taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 4 to 8 ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), or tertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³). Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃, —NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and —C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², together with the nitrogen atom to which they are attached, form a heterocyclic structure as in, for example, piperidinocarbonyl, morpholinocarbonyl, thiomorpholinocarbonyl, and piperazinocarbonyl.

Thioamido (thiocarbamyl): —C(═S)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of amido groups include, but are not limited to, —C(═S)NH₂, —C(═S)NHCH₃, —C(═S)N(CH₃)₂, and —C(═S)NHCH₂CH₃.

Acylamido (acylamino): —NR¹C(═O)R², wherein R¹ is an amide substituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group, and R² is an acyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples of acylamide groups include, but are not limited to, —NHC(═O)CH₃, —NHC(═O)CH₂CH₃, and —NHC(═O)Ph. R¹ and R² may together form a cyclic structure, as in, for example, succinimidyl, maleimidyl, and phthalimidyl:

Aminocarbonyloxy: —OC(═O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of aminocarbonyloxy groups include, but are not limited to, —OC(═O)NH₂, —OC(═O)NHMe, —OC(═O)NMe₂, and —OC(═O)NEt₂.

Ureido: —N(R¹)CONR²R³ wherein R² and R³ are independently amino substituents, as defined for amino groups, and R¹ is a ureido substituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably hydrogen or a C₁₋₇ alkyl group. Examples of ureido groups include, but are not limited to, —NHCONH₂, —NHCONHMe, —NHCONHEt, —NHCONMe₂, —NHCONEt₂, —NMeCONH₂, —NMeCONHMe, —NMeCONHEt, —NMeCONMe₂, and —NMeCONEt₂.

Guanidino: —NH—C(═NH)NH₂.

Tetrazolyl: a five membered aromatic ring having four nitrogen atoms and one carbon atom,

Imino: ═NR, wherein R is an imino substituent, for example, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇alkyl group. Examples of imino groups include, but are not limited to, ═NH, ═NMe, and ═NEt.

Amidine (amidino): —C(═NR)NR₂, wherein each R is an amidine substituent, for example, hydrogen, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably H or a C₁₋₇ alkyl group. Examples of amidine groups include, but are not limited to, —C(═NH)NH₂, —C(═NH)NMe₂, and —C(═NMe)NMe₂.

Nitro: —NO₂.

Nitroso: —NO.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Isocyano: —NC.

Cyanato: —OCN.

Isocyanato: —NCO.

Thiocyano (thiocyanato): —SCN.

Isothiocyano (isothiocyanato): —NCS.

Sulfhydryl (thiol, mercapto): —SH.

Thioether (sulfide): —SR, wherein R is a thioether substituent, for example, a C₁₋₇ alkyl group (also referred to as a C₁₋₇alkylthio group), a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of C₁₋₇ alkylthio groups include, but are not limited to, —SCH₃ and —SCH₂CH₃.

Disulfide: —SS—R, wherein R is a disulfide substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group (also referred to herein as C₁₋₇ alkyl disulfide). Examples of C₁₋₇ alkyl disulfide groups include, but are not limited to, —SSCH₃ and —SSCH₂CH₃.

Sulfine (sulfinyl, sulfoxide): —S(═O)R, wherein R is a sulfine substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfine groups include, but are not limited to, —S(═O)CH₃ and —S(═O)CH₂CH₃.

Sulfone (sulfonyl): —S(═O)₂R, wherein R is a sulfone substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group, including, for example, a fluorinated or perfluorinated C₁₋₇ alkyl group. Examples of sulfone groups include, but are not limited to, —S(═O)₂CH₃ (methanesulfonyl, mesyl), —S(═O)₂CF₃ (triflyl), —S(═O)₂CH₂CH₃ (esyl), —S(═O)₂C₄F₉ (nonaflyl), —S(═O)₂CH₂CF₃ (tresyl), —S(═O)₂CH₂CH₂NH₂ (tauryl), —S(═O)₂Ph (phenylsulfonyl, besyl), 4-methylphenylsulfonyl (tosyl), 4-chlorophenylsulfonyl (closyl), 4-bromophenylsulfonyl (brosyl), 4-nitrophenyl (nosyl), 2-naphthalenesulfonate (napsyl), and 5-dimethylamino-naphthalen-1-ylsulfonate (dansyl).

Sulfinic acid (sulfino): —S(═O)OH, —SO₂H.

Sulfonic acid (sulfo): —S(═O)₂OH, —SO₃H.

Sulfinate (sulfinic acid ester): —S(═O)OR; wherein R is a sulfinate substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinate groups include, but are not limited to, —S(═O)OCH₃ (methoxysulfinyl; methyl sulfinate) and —S(═O)OCH₂CH₃ (ethoxysulfinyl; ethyl sulfinate).

Sulfonate (sulfonic acid ester): —S(═O)₂OR, wherein R is a sulfonate substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonate groups include, but are not limited to, —S(═O)₂OCH₃ (methoxysulfonyl; methyl sulfonate) and —S(═O)₂OCH₂CH₃ (ethoxysulfonyl; ethyl sulfonate).

Sulfinyloxy: —OS(═O)R, wherein R is a sulfinyloxy substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinyloxy groups include, but are not limited to, —OS(═O)CH₃ and —OS(═O)CH₂CH₃.

Sulfonyloxy: —OS(═O)₂R, wherein R is a sulfonyloxy substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonyloxy groups include, but are not limited to, —OS(═O)₂CH₃ (mesylate) and —OS(═O)₂CH₂CH₃ (esylate).

Sulfate: —OS(═O)₂OR; wherein R is a sulfate substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfate groups include, but are not limited to, —OS(═O)₂OCH₃ and —SO(═O)₂OCH₂CH₃.

Sulfamyl (sulfamoyl; sulfinic acid amide; sulfinamide): —S(═O)NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of sulfamyl groups include, but are not limited to, —S(═O)NH₂, —S(═O)NH(CH₃), —S(═O)N(CH₃)₂, —S(═O)NH(CH₂CH₃), —S(═O)N(CH₂CH₃)₂, and —S(═O)NHPh.

Sulfonamido (sulfinamoyl; sulfonic acid amide; sulfonamide): —S(═O)₂NR¹R², wherein R¹ and R² are independently amino substituents, as defined for amino groups. Examples of sulfonamido groups include, but are not limited to, —S(═O)₂NH₂, —S(═O)₂NH(CH₃), —S(═O)₂N(CH₃)₂, —S(═O)₂NH(CH₂CH₃), —S(═O)₂N(CH₂CH₃)₂, and —S(═O)₂NHPh.

Sulfamino: —NR¹S(═O)₂OH, wherein R¹ is an amino substituent, as defined for amino groups. Examples of sulfamino groups include, but are not limited to, —NHS(═O)₂OH and —N(CH₃)S(═O)₂OH.

Sulfonamino: —NR¹S(═O)₂R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfonamino substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfonamino groups include, but are not limited to, —NHS(═O)₂CH₃ and —N(CH₃)S(═O)₂C₆H₅.

Sulfinamino: —NR¹S(═O)R, wherein R¹ is an amino substituent, as defined for amino groups, and R is a sulfinamino substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group. Examples of sulfinamino groups include, but are not limited to, —NHS(═O)CH₃ and —N(CH₃)S(═O)C₆H₅.

Phosphino (phosphine): —PR₂, wherein R is a phosphino substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphino groups include, but are not limited to, —PH₂, —P(CH₃)₂, —P(CH₂CH₃)₂, —P(t-Bu)₂, and —P(Ph)₂.

Phospho: —P(═O)₂.

Phosphinyl (phosphine oxide): —P(═O)R₂, wherein R is a phosphinyl substituent, for example, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkyl group or a C₅₋₂₀ aryl group. Examples of phosphinyl groups include, but are not limited to, —P(═O)(CH₃)₂, —P(═O)(CH₂CH₃)₂, —P(═O)(t-Bu)₂, and —P(═O)(Ph)₂.

Phosphonic acid (phosphono): —P(═O)(OH)₂.

Phosphonate (phosphono ester): —P(═O)(OR)₂, where R is a phosphonate substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphonate groups include, but are not limited to, —P(═O)(OCH₃)₂, —P(═O)(OCH₂CH₃)₂, —P(═O)(O-t-Bu)₂, and —P(═O)(OPh)₂.

Phosphoric acid (phosphonooxy): —OP(═O)(OH)₂.

Phosphate (phosphonooxy ester): —OP(═O)(OR)₂, where R is a phosphate substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphate groups include, but are not limited to, —OP(═O)(OCH₃)₂, —OP(═O)(OCH₂CH₃)₂, —OP(═O)(O-t-Bu)₂, and —OP(═O)(OPh)₂.

Phosphorous acid: —OP(OH)₂.

Phosphite: —OP(OR)₂, where R is a phosphite substituent, for example, —H, a C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphite groups include, but are not limited to, —OP(OCH₃)₂, —OP(OCH₂CH₃)₂, —OP(O-t-Bu)₂, and —OP(OPh)₂.

Phosphoramidite: —OP(OR¹)—NR²², where R¹ and R² are phosphoramidite substituents, for example, —H, a (optionally substituted) C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidite groups include, but are not limited to, —OP(OCH₂CH₃)—N(CH₃)₂, —OP(OCH₂CH₃)—N(i-Pr)₂, and —OP(OCH₂CH₂CN)—N(i-Pr)₂.

Phosphoramidate: —OP(═O)(OR¹)—NR²², where R¹ and R² are phosphoramidate substituents, for example, —H, a (optionally substituted) C₁₋₇ alkyl group, a C₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably —H, a C₁₋₇ alkyl group, or a C₅₋₂₀ aryl group. Examples of phosphoramidate groups include, but are not limited to, —OP(═O)(OCH₂CH₃)—N(CH₃)₂, —OP(═O)(OCH₂CH₃)—N(i-Pr)₂, and —OP(═O)(OCH₂CH₂CN)—N(i-Pr)₂.

Alkylene

C₃₋₁₂ alkylene: The term “C₃₋₁₂ alkylene”, as used herein, pertains to a bidentate moiety obtained by removing two hydrogen atoms, either both from the same carbon atom, or one from each of two different carbon atoms, of a hydrocarbon compound having from 3 to 12 carbon atoms (unless otherwise specified), which may be aliphatic or alicyclic, and which may be saturated, partially unsaturated, or fully unsaturated. Thus, the term “alkylene” includes the sub-classes alkenylene, alkynylene, cycloalkylene, etc., discussed below.

Examples of linear saturated C₃₋₁₂ alkylene groups include, but are not limited to, —(CH₂)_(n)— where n is an integer from 3 to 12, for example, —CH₂CH₂CH₂— (propylene), —CH₂CH₂CH₂CH₂— (butylene), —CH₂CH₂CH₂CH₂CH₂— (pentylene) and —CH₂CH₂CH₂CH—₂CH₂CH₂CH₂— (heptylene).

Examples of branched saturated C₃₋₁₂alkylene groups include, but are not limited to, —CH(CH₃)CH₂—, —CH(CH₃)CH₂CH₂—, —CH(CH₃)CH₂CH₂CH₂—, —CH₂CH(CH₃)CH₂—, —CH₂CH(CH₃)CH₂CH₂—, —CH(CH₂CH₃)—, —CH(CH₂CH₃)CH₂—, and —CH₂CH(CH₂CH₃)CH₂—.

Examples of linear partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂ alkenylene, and alkynylene groups) include, but are not limited to, —CH═CH—CH₂—, —CH₂—CH═CH₂—, —CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH₂—CH₂—, —CH═CH—CH═CH—, —CH═CH—CH═CH—CH₂—, —CH═CH—CH═CH—CH₂—CH₂—, —CH═CH—CH₂—CH═CH—, —CH═CH—CH₂—CH₂—CH═CH—, and —CH₂—C≡C—CH₂—.

Examples of branched partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂ alkenylene and alkynylene groups) include, but are not limited to, —C(CH₃)═CH—, —C(CH₃)═CH—CH₂—, —CH═CH—CH(CH₃)— and —C≡C—CH(CH₃)—.

Examples of alicyclic saturated C₃₋₁₂ alkylene groups (C₃₋₁₂ cycloalkylenes) include, but are not limited to, cyclopentylene (e.g. cyclopent-1,3-ylene), and cyclohexylene (e.g. cyclohex-1,4-ylene).

Examples of alicyclic partially unsaturated C₃₋₁₂ alkylene groups (C₃₋₁₂ cycloalkylenes) include, but are not limited to, cyclopentenylene (e.g. 4-cyclopenten-1,3-ylene), cyclohexenylene (e.g. 2-cyclohexen-1,4-ylene; 3-cyclohexen-1,2-ylene; 2,5-cyclohexadien-1,4-ylene).

Includes Other Forms

Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (—COOH) also includes the anionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (—N⁺HR¹R²), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (—O⁻), a salt or solvate thereof, as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66, 1-19 (1977).

For example, if the compound is anionic, or has a functional group which may be anionic (e.g. —COOH may be —COO⁻), then a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may be cationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acid and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the active compound. The term “solvate” is used herein in the conventional sense to refer to a complex of solute (e.g. active compound, salt of active compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

The invention includes compounds where a solvent adds across the imine bond of the PBD moiety, which is illustrated below where the solvent is water or an alcohol (R^(A)OH, where R^(A) is C₁₋₄ alkyl):

These forms can be called the carbinolamine and carbinolamine ether forms of the PBD (as described in the section relating to R¹⁰ above). The balance of these equilibria depend on the conditions in which the compounds are found, as well as the nature of the moiety itself.

These particular compounds may be isolated in solid form, for example, by lyophilisation.

Isomers

Certain compounds of the invention may exist in one or more particular geometric, optical, enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as “isomers” (or “isomeric forms”).

The term “chiral” refers to molecules which have the property of non-superimposability of the mirror image partner, while the term “achiral” refers to molecules which are superimposable on their mirror image partner.

The term “stereoisomers” refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of one another. Diastereomers have different physical properties, e.g. melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may separate under high resolution analytical procedures such as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which are non-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., New York, 1994. The compounds of the invention may contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds of the invention, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and I or (+) and (−) are employed to designate the sign of rotation of plane-polarized light by the compound, with (−) or l meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which may occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms “racemic mixture” and “racemate” refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

Note that, except as discussed below for tautomeric forms, specifically excluded from the term “isomers”, as used herein, are structural (or constitutional) isomers (i.e. isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, —OCH₃, is not to be construed as a reference to its structural isomer, a hydroxymethyl group, —CH₂OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. However, a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C₁₋₇ alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

The term “tautomer” or “tautomeric form” refers to structural isomers of different energies which are interconvertible via a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include interconversions via migration of a proton, such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions by reorganization of some of the bonding electrons.

Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including ¹H, ²H (D), and ³H (T); C may be in any isotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopic form, including ¹⁶O and ¹⁸O; and the like.

Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, and chlorine, such as, but not limited to ²H (deuterium, D), ³H (tritium), ¹¹C, ¹³C ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S, ³⁶Cl, and ¹²⁵I. Various isotopically labeled compounds of the present invention, for example those into which radioactive isotopes such as 3H, 13C, and 14C are incorporated. Such isotopically labelled compounds may be useful in metabolic studies, reaction kinetic studies, detection or imaging techniques, such as positron emission tomography (PET) or single-photon emission computed tomography (SPECT) including drug or substrate tissue distribution assays, or in radioactive treatment of patients. Deuterium labelled or substituted therapeutic compounds of the invention may have improved DMPK (drug metabolism and pharmacokinetics) properties, relating to distribution, metabolism, and excretion (ADME). Substitution with heavier isotopes such as deuterium may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements. An 18F labeled compound may be useful for PET or SPECT studies. Isotopically labeled compounds of this invention and prodrugs thereof can generally be prepared by carrying out the procedures disclosed in the schemes or in the examples and preparations described below by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent. Further, substitution with heavier isotopes, particularly deuterium (i.e., 2H or D) may afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo half-life or reduced dosage requirements or an improvement in therapeutic index. It is understood that deuterium in this context is regarded as a substituent. The concentration of such a heavier isotope, specifically deuterium, may be defined by an isotopic enrichment factor. In the compounds of this invention any atom not specifically designated as a particular isotope is meant to represent any stable isotope of that atom.

Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof. Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Biological Activity

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of an antibody-drug conjugate (ADC) is measured by: exposing mammalian cells having receptor proteins, e.g. HER2, to the antibody of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 5 days; and measuring cell viability. Cell-based in vitro assays are used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of an ADC of the invention.

The in vitro potency of antibody-drug conjugates can be measured by a cell proliferation assay. The CellTiter-Glo® Luminescent Cell Viability Assay is a commercially available (Promega Corp., Madison, Wis.), homogeneous assay method based on the recombinant expression of Coleoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713 and 5,700,670). This cell proliferation assay determines the number of viable cells in culture based on quantitation of the ATP present, an indicator of metabolically active cells (Crouch et al (1993) J. Immunol. Meth. 160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay is conducted in 96 well format, making it amenable to automated high-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs 6:398-404). The homogeneous assay procedure involves adding the single reagent (CellTiter-Glo® Reagent) directly to cells cultured in serum-supplemented medium. Cell washing, removal of medium and multiple pipetting steps are not required. The system detects as few as 15 cells/well in a 384-well format in 10 minutes after adding reagent and mixing. The cells may be treated continuously with ADC, or they may be treated and separated from ADC. Generally, cells treated briefly, i.e. 3 hours, showed the same potency effects as continuously treated cells.

The homogeneous “add-mix-measure” format results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. The CellTiter-Glo® Assay generates a “glow-type” luminescent signal, produced by the luciferase reaction, which has a half-life generally greater than five hours, depending on cell type and medium used. Viable cells are reflected in relative luminescence units (RLU). The substrate, Beetle Luciferin, is oxidatively decarboxylated by recombinant firefly luciferase with concomitant conversion of ATP to AMP and generation of photons.

The in vitro potency of antibody-drug conjugates can also be measured by a cytotoxicity assay. Cultured adherent cells are washed with PBS, detached with trypsin, diluted in complete medium, containing 10% FCS, centrifuged, re-suspended in fresh medium and counted with a haemocytometer. Suspension cultures are counted directly. Monodisperse cell suspensions suitable for counting may require agitation of the suspension by repeated aspiration to break up cell clumps.

The cell suspension is diluted to the desired seeding density and dispensed (100 μl per well) into black 96 well plates. Plates of adherent cell lines are incubated overnight to allow adherence. Suspension cell cultures can be used on the day of seeding.

A stock solution (1 ml) of ADC (20 μg/ml) is made in the appropriate cell culture medium. Serial 10-fold dilutions of stock ADC are made in 15 ml centrifuge tubes by serially transferring 100 μl to 900 μl of cell culture medium.

Four replicate wells of each ADC dilution (100 μl) are dispensed in 96-well black plates, previously plated with cell suspension (100 μl), resulting in a final volume of 200 μl. Control wells receive cell culture medium (100 μl).

If the doubling time of the cell line is greater than 30 hours, ADC incubation is for 5 days, otherwise a four day incubation is done.

At the end of the incubation period, cell viability is assessed with the Alamar blue assay. AlamarBlue (Invitrogen) is dispensed over the whole plate (20 μl per well) and incubated for 4 hours. Alamar blue fluorescence is measured at excitation 570 nm, emission 585 nm on the Varioskan flash plate reader. Percentage cell survival is calculated from the mean fluorescence in the ADC treated wells compared to the mean fluorescence in the control wells.

In Vivo Efficacy

The in vivo efficacy of antibody-drug conjugates (ADC) of the invention can be measured by tumor xenograft studies in mice. For example, the in vivo efficacy of an anti-HER2 ADC of the invention can be measured by a high expressing HER2 transgenic explant mouse model. An allograft is propagated from the Fo5 mmtv transgenic mouse which does not respond to, or responds poorly to, HERCEPTIN® therapy. Subjects were treated once with ADC at certain dose levels (mg/kg) and PBD drug exposure (μg/m²); and placebo buffer control (Vehicle) and monitored over two weeks or more to measure the time to tumor doubling, log cell kill, and tumor shrinkage.

Use

The conjugates of the invention may be used to provide a PBD conjugate at a target location.

The target location is preferably a proliferative cell population. The antibody is an antibody for an antigen present in a proliferative cell population.

In one embodiment the antigen is absent or present at a reduced level in a non-proliferative cell population compared to the amount of antigen present in the proliferative cell population, for example a tumour cell population.

The target location may be in vitro, in vivo or ex vivo.

The antibody-drug conjugate (ADC) compounds of the invention include those with utility for anticancer activity. In particular, the compounds include an antibody conjugated, i.e. covalently attached by a linker, to a PBD moiety.

At the target location the linker may be cleaved. The antibody-drug conjugate (ADC compounds of the invention may have a cytotoxic effect with the cleavage of the linker to release a PBD drug moiety. The antibody-drug conjugates (ADC) of the invention selectively deliver cytotoxic agent to tumor tissue whereby greater selectivity, i.e. a lower efficacious dose, may be achieved.

Thus, in one aspect, the present invention provides a conjugate compound as described herein for use in therapy.

In a further aspect there is also provides a conjugate compound as described herein for use in the treatment of a proliferative disease. A second aspect of the present invention provides the use of a conjugate compound in the manufacture of a medicament for treating a proliferative disease.

One of ordinary skill in the art is readily able to determine whether or not a candidate conjugate treats a proliferative condition for any particular cell type. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described in the examples below.

The term “proliferative disease” pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.

Examples of proliferative conditions include, but are not limited to, benign, pre-malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g. histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Cancers of particular interest include, but are not limited to, leukemias and ovarian cancers.

Any type of cell may be treated, including but not limited to, lung, gastrointestinal (including, e.g. bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.

In one embodiment, the treatment is of a pancreatic cancer.

In one embodiment, the treatment is of a tumour having α_(v)β₆ integrin on the surface of the cell.

It is contemplated that the antibody-drug conjugates (ADC) of the present invention may be used to treat various diseases or disorders, e.g. characterized by the overexpression of a tumor antigen. Exemplary conditions or hyperproliferative disorders include benign or malignant tumors; leukemia, haematological, and lymphoid malignancies. Others include neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic, including autoimmune, disorders.

Generally, the disease or disorder to be treated is a hyperproliferative disease such as cancer. Examples of cancer to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.

Autoimmune diseases for which the ADC compounds may be used in treatment include rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g. ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteriitis), autoimmune neurological disorders (such as, for example, multiple sclerosis, opsoclonus myoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathies), renal disorders (such as, for example, glomerulonephritis, Goodpasture's syndrome, and Berger's disease), autoimmune dermatologic disorders (such as, for example, psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), hematologic disorders (such as, for example, thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as, for example, inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as, for example, diabetic-related autoimmune diseases such as insulin-dependent diabetes mellitus (IDDM), Addison's disease, and autoimmune thyroid disease (e.g. Graves' disease and thyroiditis)). More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.

Methods of Treatment

The conjugates of the present invention may be used in a method of therapy. Also provided is a method of treatment, comprising administering to a subject in need of treatment a therapeutically-effective amount of a conjugate compound of the invention. The term “therapeutically effective amount” is an amount sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage, is within the responsibility of general practitioners and other medical doctors.

A compound of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs, such as chemotherapeutics); surgery; and radiation therapy.

A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer, regardless of mechanism of action. Classes of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, spindle poison plant alkaloids, cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in “targeted therapy” and conventional chemotherapy.

Examples of chemotherapeutic agents include: erlotinib (TARCEVA®, Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®, Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin (cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0] nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®, Schering Plough), tamoxifen ((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine, NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2, HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin (ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent (SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO 2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH 66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11, Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™ (Cremophor-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, II), vandetanib (rlNN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib (GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa and cyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analog topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, calicheamicin gammall, calicheamicin omegall (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are: (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those which inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitors such as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptable salts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential as chemotherapeutic agents in combination with the conjugates of the invention include: alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and visilizumab.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to the active ingredient, i.e. a conjugate compound, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. A capsule may comprise a solid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Formulations

While it is possible for the conjugate compound to be used (e.g., administered) alone, it is often preferable to present it as a composition or formulation.

In one embodiment, the composition is a pharmaceutical composition (e.g., formulation, preparation, medicament) comprising a conjugate compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.

In one embodiment, the composition is a pharmaceutical composition comprising at least one conjugate compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other active agents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts. See, for example, Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott, N.Y., USA), Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott, Williams & Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition, 1994.

Another aspect of the present invention pertains to methods of making a pharmaceutical composition comprising admixing at least one [¹¹C]-radiolabelled conjugate or conjugate-like compound, as defined herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.

The term “pharmaceutically acceptable,” as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.

The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.

Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active ingredient in the liquid is from about 1 ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriate dosages of the conjugate compound, and compositions comprising the conjugate compound, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range of about 100 ng to about 25 mg (more typically about 1 μg to about 10 mg) per kilogram body weight of the subject per day. Where the active compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.

In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 100 mg, 3 times daily.

In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 150 mg, 2 times daily.

In one embodiment, the active compound is administered to a human patient according to the following dosage regime: about 200 mg, 2 times daily.

However in one embodiment, the conjugate compound is administered to a human patient according to the following dosage regime: about 50 or about 75 mg, 3 or 4 times daily.

In one embodiment, the conjugate compound is administered to a human patient according to the following dosage regime: about 100 or about 125 mg, 2 times daily.

The dosage amounts described above may apply to the conjugate (including the PBD moiety and the linker to the antibody) or to the effective amount of PBD compound provided, for example the amount of compound that is releasable after cleavage of the linker.

For the prevention or treatment of disease, the appropriate dosage of an ADC of the invention will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the molecule is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. An exemplary dosage of ADC to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. An exemplary dosing regimen comprises a course of administering an initial loading dose of about 4 mg/kg, followed by additional doses every week, two weeks, or three weeks of an ADC. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.

Treatment

The term “treatment,” as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, regression of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

Similarly, the term “prophylactically-effective amount,” as used herein, pertains to that amount of an active compound, or a material, composition or dosage from comprising an active compound, which is effective for producing some desired prophylactic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.

Preparation of Antibody Drug Conjugates

Antibody drug conjugates may be prepared by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group of an antibody with a bivalent linker reagent, to form antibody-linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated drug moiety reagent; and (2) reaction of a drug moiety reagent with a linker reagent, to form drug-linker reagent D-L, via a covalent bond, followed by reaction with the nucleophilic of an antibody. Conjugation methods (1) and (2) may be employed with a variety of antibodies, and linkers to prepare the antibody-drug conjugates of the invention.

Nucleophilic groups on antibodies include, but are not limited to side chain thiol groups, e.g. cysteine. Thiol groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties such as those of the present invention. Certain antibodies have reducible interchain disulfides, i.e. cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (Cleland's reagent, dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal. Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.). Each cysteine disulfide bridge will thus form, theoretically, two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.

The Subject/Patient

The subject/patient may be an animal, mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilled platypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang, gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development, for example, a foetus. In one preferred embodiment, the subject/patient is a human. In one embodiment, the patient is a population where each patient has a tumour having α_(v)β₆ integrin on the surface of the cell.

Synthesis

Conjugates of formula A may be synthesised from corresponding drug-linker compounds of formula B by reacting them with cell binding agents under appropriate conditions. Thus, conjugates where Y is of formula A1 may be synthesised from drug-linker compounds where Y^(L) is of formula B1. Conjugates where Y is of formula A2 may be synthesised from drug-linker compounds where Y^(L) is of formula B2.

The conditions, as described above, will depend on the type of bond being formed between the drug-linker compound and the cell binding agent, which itself will reflect the nature of the binding site on the cell binding agent.

Drug-linker compounds of formula B may be synthesised from corresponding compounds of formula C.

Drug-linker compounds where Y^(L) is of formula B1 may be synthesised from compounds where Y^(C) is of formula C1, by reaction with a compound of formula E1:

in an appropriate solvent, where Q′ is

-   -    in the presence of an amide coupling reagent.

Drug-linker compounds where Y^(L) is of formula B2 may be synthesised from compounds where Y^(C) is of formula C2, by reaction with a compound of formula E1 in an appropriate solvent, in the presence of an amide coupling reagent.

Alternatively, the group formed by E1 may be added in two steps, adding compound F1 (or a protected version thereof) in an appropriate solvent, in the presence of an amide coupling reagent, followed by F2 in an appropriate solvent, in the presence of an amide coupling reagent. If a protected version of F1 is used, it will be deprotected before F2 is added.

Compound of formula C can be made from the corresponding compound of formula G:

Compounds of formula C where Y^(C) is of formula C1 may be synthesised by reacting a compound of formula G with a compound of formula H1:

where Prot^(N) is an amine protecting group, such as Alloc, in the presence of tetrabutylammonium iodide and potassium carbonate, followed by deprotection of the amine under standard conditions. The protecting group used should be orthogonal to any other protecting groups in the compound.

Compounds of formula C where Y^(C) is of formula C2 may be synthesised by reacting a compound of formula G with a compound of formula H2:

where Prot^(N) is an amine protecting group, such as Alloc, in the presence of tetrabutylammonium iodide and potassium carbonate, followed by deprotection of the amine under standard conditions. The protecting group used should be orthogonal to any other protecting groups in the compound.

Alternatively, compounds of formula C where Y^(C) is of formula C2 may be synthesised by reacting a compound of formula G with a compound of formula H3:

in the presence of tetrabutylammonium iodide and potassium carbonate, followed by reduction of the nitro group under standard conditions.

Compound of formula G containing a single PBD moiety can be synthesised according to the disclosure of WO 2005/085259, and in particular the discussion from pages 31 to 39, which is incorporated herein be reference. Reference is also made to the teaching of co-pending application PCT/EP2012/070232, filed on 12 Oct. 2012.

The synthesis of PBD compounds containing two imine moieties is extensively discussed in the following references, which discussions are incorporated herein by reference:

a) WO 00/12508 (pages 14 to 30);

b) WO 2005/023814 (pages 3 to 10);

c) WO 2004/043963 (pages 28 to 29);

d) WO 2005/085251 (pages 30 to 39); and

e) WO 2011/130598 (pages 126 to 150).

The disclosure of WO 2005/085259 discussed above is also relevant to the synthesis of compounds of formula G which comprise two PBD moieties. The synthesis methods disclosed therein may be modified to include an orthogonally protected hydroxyl group at C7 (i.e. group R^(A) in Scheme 4).

Alternatively, compounds of formula G may be synthesised as described in the above references, but starting from a dimer core of formula J:

where Prot^(O) is a hydroxyl protecting group. Such compounds of formula J may be made by methods analogous to those in the examples of the present application.

In other methods, compounds of K1 or K2:

may be reacted with G, followed by conversion to drug linkers of formula B, by deprotection and addition of a compound of formula F2. Amine Protecting Groups

Amine protecting groups are well-known to those skilled in the art. Particular reference is made to the disclosure of suitable protecting groups in Greene's Protecting Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages 696-871.

Hydroxyl Protecting Groups

Hydroxyl protecting groups are well-known to those skilled in the art. Particular reference is made to the disclosure of suitable protecting groups in Greene's Protecting Groups in Organic Synthesis, Fourth Edition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages 16-298.

EXAMPLES

General Conditions

Reaction progress was monitored by thin-layer chromatography (TLC) using Merck Kieselgel 60 F254 silica gel, with fluorescent indicator on aluminium plates. Visualisation of TLC was achieved with UV light or iodine vapour unless otherwise stated. Flash chromatography was performed using Merck Kieselgel 60 F254 silica gel. Extraction and chromatography solvents were bought and used without further purification from Fisher Scientific, U.K. All chemicals were purchased from Aldrich, Lancaster or BDH.

¹H and ¹³C NMR spectra were obtained on a Bruker Avance 400 spectrometer. Coupling constants are quoted in hertz (Hz). Chemical shifts are recorded in parts per million (ppm) downfield from tetramethylsilane. Spin multiplicities are described as s (singlet), bs (broad singlet), d (doublet), t (triplet), q (quartet), p (pentuplet) and m (multiplet).

LCMS Method

The HPLC (Shimazu LCMS-2020) was run using a mobile phase of water (A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%). Gradient: initial composition 5% B held over 0.25 min, then increase from 5% B to 100% B over a 2 min period. The composition was held for 0.50 min at 100% B, then returned to 5% B in 0.05 minutes and hold there for 0.05 min. Total gradient run time equals 3 minutes.

Flow rate 0.8 mL/min. Wavelength detection range: 220 to 400 nm. Column: Waters Acquity UPLC BEH Shield RP18 1.7 μm 2.1×50 mm.

Synthesis of Key Intermediates (a) 5-(benzyloxy)-4-((5-(4-carboxy-2-methoxy-5-nitrophenoxy)pentyl)oxy)-2-nitrobenzoic acid (I7)

(i) 3-(benzyloxy)-4-hydroxybenzaldehyde (I2)

Sodium hydride (51.2 g, 1.27 mol, 2.2 eq) was rinsed twice with hexane in a three-neck flask and anhydrous DMSO (800 mL) was added. The flask was placed in a water bath at room temperature. A solution of 3,4-dihydroxybenzaldehyde I1 (80 g, 579.2 mmol) in dry DMSO (160 mL) was added dropwise, with an addition funnel, over 40 minutes and the reaction mixture was stirred 30 minutes under Argon. During the addition a lot of hydrogen gas is formed, so an exit with cotton wool and calcium chloride is placed in one of the neck. Benzyl bromide (68.8 mL, 579.2 mmol, 1 eq) was then added dropwise and the reaction was stirred overnight. The reaction mixture was poured into ice and quenched with HCl (1M) until pH acid and extracted with EtOAc. The organic phase was then washed with brine and was dried over magnesium sulphate, filtered and excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to a pad of Silica with pure dichloromethane. The resulting material was precipitated with a minimum of dichloromethane in hexane. The resulting white precipitate was filtered and dried to afford the desired compound (80.7 g, 60% yield). LC/MS 1.43 min (no ionisation). ¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, 1H), 7.51 (d, J=1.8 Hz, 1H), 7.45-7.34 (m, 7H), 7.06 (d, J=8.1 Hz, 1H), 6.24 (s, 1H), 5.17 (s, 2H).

(ii) 4-((5-bromopentyl)oxy)-3-methoxybenzaldehyde (I4)

Vanillin I3 (50 g, 328 mmol) was dissolved in acetone (1 L). Dibromopentane (227 g, 985 mmol, 3 eq) and potassium carbonate (68 g, 492 mmol, 1.5 eq) were added. The slurry was warmed to 65° C. and stirred for 2 hours, and then 80° C. for 30 minutes. The resulting potassium carbonate was filtered and the excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to a pad of Silica: 7.5% EtOAc in Hexane (4 L) then 10% EtOAc in Hexane (1 L) and 25% EtOAc in Hexane to afford a white solid (53.7 g, 54% yield). LC/MS 1.63 min (ES+) m/z (relative intensity) 302.53 ([M+H]⁺, 100). ¹H NMR (400 MHz, CDCl₃) δ 9.83 (s, 1H), 7.42 (dd, J=8.1, 1.9 Hz, 1H), 7.40 (d, J=1.9 Hz, 1H), 6.95 (d, J=8.1 Hz, 1H), 4.10 (t, J=6.6 Hz, 2H), 3.91 (s, 3H), 3.43 (t, J=6.7 Hz, 2H), 1.92 (m, 4H), 1.64 (tt, J=9.7, 6.2 Hz, 2H).

(iii) 3-(benzyloxy)-4-((5-(4-formyl-2-ethoxyphenoxy)pentyl)oxy) benzaldehyde (I5)

4-((5-bromopentyl)oxy)-3-methoxybenzaldehyde I4 (40.0 g, 132.81 mmol, 1 eq) and 3-(benzyloxy)-4-hydroxybenzaldehyde I2 (30.3 g, 132.81 mmol, 1 eq) were dissolved in dimethylformamide (200 mL). Potassium carbonate (13.8 g, 99.61 mmol, 0.75 eq) and tetrabutylammonium iodide (4.9 g, 13.28 mmol, 0.1 eq) were added. The reaction mixture was warmed to 80° C. and stirred for 12 hours. The resulting potassium carbonate was filtered and the excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was dissolved in Ethyl acetate and washed subsequently with water, 1N NaOH, 1N HCl, water and brine. The organic phase was dried over magnesium sulphate, filtered and excess solvent was removed by rotary evaporation under reduced pressure to afford the desired compound (75 g, quantitative yield) as a light yellow oil. LC/MS 1.77 min (ES+) m/z (relative intensity) 449.15 [M+H]⁺, 471.25 [M+Na]⁺. ¹H NMR (400 MHz, CDCl₃) δ 9.83 (s, 1H), 9.81 (s, 1H), 7.46 (dd, J=3.1, 1.4 Hz, 2H), 7.43 (d, J=1.7 Hz, 2H), 7.41 (dd, J=8.1, 1.9 Hz, 1H), 7.39 (d, J=1.8 Hz, 1H), 7.37-7.32 (m, 2H), 7.30 (dd, J=5.0, 3.6 Hz, 1H), 6.97 (d, J=8.1 Hz, 1H), 6.92 (d, J=8.1 Hz, 1H), 5.15 (s, 2H), 4.13 (t, J=6.4 Hz, 2H), 4.09 (t, J=6.6 Hz, 2H), 3.88 (s, 3H), 2.01-1.89 (m, 4H), 1.77-1.64 (m, 2H).

(iv) 5-(benzyloxy)-4-((5-(4-formyl-2-methoxy-5-nitrophenoxy)pentyl)oxy)-2-nitrobenzaldehyde (I6)

3-(benzyloxy)-4-((5-(4-formyl-2-ethoxyphenoxy)pentyl)oxy) benzaldehyde I5 (30 g, 66.40 mmol) was dissolved in dichloromethane (60 mL) and added to nitric acid (68%, 60 mL) at 0° C. The reaction mixture was stirred at 0° C. for 20 minutes and at room temperature for 3 hours. The reaction mixture gets darker after 1 hour. After 2 hours, the LCMS showed no more starting material, so cold water was added and the precipitate was filtered and washed with water. The white solid was dried by vacuum. 42 g of white solid was obtained (>100% yield due to water remaining). LC/MS 1.88 min, no ionisation. ¹H NMR (400 MHz, CDCl₃) δ 10.44 (s, 1H), 10.41 (s, 1H), 7.61 (s, 1H), 7.56 (s, 1H), 7.48 (s, 1H), 7.46-7.30 (m, 6H), 5.25 (s, 2H), 4.20 (dd, J=12.2, 5.9 Hz, 2H), 4.13 (dd, J=15.1, 8.7 Hz, 2H), 3.96 (s, 3H), 2.01 (dt, J=14.1, 6.4 Hz, 4H), 1.80-1.65 (m, 2H).

(v) 5-(benzyloxy)-4-((5-(4-carboxy-2-methoxy-5-nitrophenoxy)pentyl)oxy)-2-nitrobenzoic acid (I7)

A solution of sodium chlorite (35.3 g, 390 mmol, 5 eq) and sodium phosphate (26.2 g, 218.4 mmol, 2.8 eq) in water (250 mL) was added to a solution of 5-(benzyloxy)-4-((5-(4-formyl-2-methoxy-5-nitrophenoxy)pentyl)oxy)-2 nitrobenzaldehyde I6 (42 g, 78 mmol) in THF (200 mL). Hydrogen peroxide (60%, 103 mL, 2.18 mol, 28 eq) was then added quickly. No more starting material was observed after 30 minutes by LCMS/TLC. The reaction was then quenched with 1N HCl and extracted with ethyl acetate. The organic layer was washed two times with brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was dissolved in a minimum of dichloromethane and precipitated out with ether. Pale yellow solid (26 g, 58%) was filtered and washed with ether and used as crude for the next reaction.

LC/MS 1.69 min (ES+) m/z (relative intensity) 569.35 [M+H]⁺.

(b) (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-5,11-dioxo-7-((triisopropylsilyl)oxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methy)-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (I13)

(i) (2S,4R)-methyl 1-(4-((5-(2-(benzyloxy)-4-((2S,4R)-4-hydroxy-2-(methoxycarbonyl)pyrrolidine-1-carbonyl)-5-nitrophenoxy)pentyl)oxy)-5-methoxy-2-nitrobenzoyl)-4-hydroxypyrrolidine-2-carboxylate (I9)

Oxalyl chloride (7.6 mL, 89.92 mmol, 3 eq) was added to a solution of I7 (17.1 g, 29.97 mmol) in dichloromethane (150 mL) and DMF (2 mL). After 20 min, a sample is taken and a small workup with MeOH is done. LCMS of this sample showed only one pic at 1.87 min and no starting material. The solvent was solvent was removed by rotary evaporation under reduced pressure and minimum of dichloromethane was added to dissolve the crude and triturated with diethyl ether. The yellow solid formed was filtered and added portion wise to a solution of 10 (13.6 g, 74.93 mmol, 2.5 eq) and triethylamine (20.92 mL, 149.87 mmol, 5 eq) in dichloromethane (100 mL) at −40° C. The reaction was complete after few minutes. The solvent was removed by rotary evaporation under reduced pressure and the resulting residue was subjected to flash column chromatography (silica gel; 50% ethyl acetate in hexane to 100% ethyl acetate to collect the mono addition product and then 5% to 20% methanol in dichloromethane). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product (15 g, 61% over 3 steps). LC/MS 1.47 min (ES+) m/z (relative intensity) 825.45 ([M+H]⁺, 100). ¹H NMR (400 MHz, CDCl₃) δ 7.65 (s, 1H), 7.61 (s, 1H), 7.45-7.27 (m, 5H), 6.89 (s, 1H), 6.81 (s, 1H), 5.20 (s, 2H), 4.85-4.74 (m, 2H), 4.40 (d, J=20.3 Hz, 2H), 4.21-4.02 (m, 4H), 3.91 (s, 3H), 3.79 (s, 6H), 3.53-3.41 (m, 2H), 3.13 (d, J=11.1 Hz, 1H), 3.04 (d, J=11.0 Hz, 1H), 2.79-2.72 (m, J=4.3 Hz, 1H), 2.63 (s, 1H), 2.42-2.32 (m, 2H), 2.21-2.06 (m, J=11.3 Hz, 2H), 2.02-1.89 (m, 4H), 1.76-1.65 (m, 2H).

(ii) (2S,4R)-methyl 1-(4-((5-(2-(benzyloxy)-4-((2S,4R)-4-((tert-butyldimethylsilyl)oxy)-2-(methoxycarbonyl)pyrrolidine-1-carbonyl)-5-nitrophenoxy)pentyl)oxy)-5-methoxy-2-nitrobenzoyl)-4-((tert-butyldimethylsilyl)oxy)pyrrolidine-2-carboxylate (I10)

I9 (14 g, 16.97 mmol), tert-butyldimethylsilyl chloride (12.8 g, 84.87 mmol, 5 eq) and imidazole (13.9 g, 203.64 mmol, 12 eq) were melted together at 120° C. (a drop of dichloromethane was added to mix the different solids on the side of the flask). The reaction was complete after 30 minutes. The resulting residue was subjected to flash column chromatography (silica gel; 10% ethyl acetate in hexane to 100% ethyl acetate). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product (17 g, quantitative).

LC/MS 2.17 min. ¹H NMR (400 MHz, CDCl₃) δ 7.69 (s, 1H), 7.65 (s, 1H), 7.43-7.27 (m, 5H), 6.87 (s, 1H), 6.78 (s, 1H), 5.17 (d, J=5.6 Hz, 2H), 4.73-7.79 (m, 2H), 4.45-4.34 (m, 2H), 4.32-4.10 (m, 2H), 4.09-4.00 (m, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 3.79 (s, 3H), 3.48-3.41 (m, 1H), 3.29 (dd, J=10.3, 4.6 Hz, 1H), 3.03 (dd, J=10.4, 2.4 Hz, 2H), 2.96 (dd, J=10.2, 2.8 Hz, 2H), 2.31-2.21 (m, 2H), 2.19-2.05 (m, 2H), 2.00-1.88 (m, 4H), 1.74-1.66 (m, 2H), 0.82 (s, 18H), 0.02 (d, J=1.1 Hz, 6H), −0.03 (d, J=5.5 Hz, 6H).

(iii) (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-7-hydroxy-5,11-dioxo-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (I11)

Ammonium formate (106 g, 168 mmol, 10 eq) was added to a solution of I10 (17.7 g, 16.80 mmol) in ethanol (500 mL). Palladium on carbon (1.8 g, 10%) was wetted with ethyl acetate and added to the reaction mixture. The solution was warmed to 70° C. (at 69° C., yellow then white gas formed). Termination of the evolution of gas marked the completion of the reaction (around 20 min). The reaction mixture was then filtered through celite and washed with ethyl acetate. The solvent was removed by rotary evaporation. The residue was dissolved in ethyl acetate and washed with saturated aqueous ammonium chloride and brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure to give the desired compound (13.9 g, 98%) as a yellow gum.

LC/MS 1.91 min, (ES+) m/z (relative intensity) 839.35 ([M+H]⁺, 100).

(iv) (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-5,11-dioxo-7-((triisopropylsilyl)oxy)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (I12)

I11 (13.9 g, 16.56 mmol), triisopropylsilyl chloride (3.6 mL, 18.36 mmol, 1.1 eq) and imidazole (3.4 g, 49.94 mmol, 3 eq) were melted together at 120° C. (a drop of dichloromethane was added to mix the different solids on the side of the flask). The reaction was complete after 30 minutes. The resulting residue was subjected to flash column chromatography (silica gel; 10% ethyl acetate in hexane to 100% ethyl acetate). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product (15.4 g, 93%). LC/MS 2.31 min, ¹H NMR (400 MHz, CDCl₃) δ 9.81 (s, 1H), 8.70 (s, 1H), 8.65 (s, 1H), 7.43 (s, 2H), 6.47 (s, 1H), 6.42 (s, 1H), 4.54-4.43 (m, 2H), 4.17 (dt, J=7.6, 3.8 Hz, 2H), 4.00 (t, J=6.4 Hz, 2H), 3.95 (t, J=6.2 Hz, 2H), 3.87 (s, 3H), 3.73-3.58 (m, 4H), 2.84-2.76 (m, 2H), 2.09-1.96 (m, 1H), 1.92-1.85 (m, 4H), 1.68-1.62 (m, 2H), 1.31-1.17 (m, 3H), 1.08 (d, J=2.5 Hz, 9H), 1.06 (d, J=2.5 Hz, 9H), 0.85 (s, 9H), 0.84 (s, 9H), 0.06 (s, 6H), 0.07 (s, 6H).

(v) (2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-8-((5-(((2R,11aS)-2-((tert-butyldimethylsilyl)oxy)-5,11-dioxo-7-((triisopropylsilyl)oxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)-7-methoxypentyl)oxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (I13)

I12 (15.4 g, 15.74 mmol) was dissolved in dry tetrahydrofuran (250 mL) and cooled to −30° C. (dry ice/acetone). n-Butyllithium (29 mL, 46.41 mmol, 3 eq) was then added dropwise and the reaction mixture was stirred for 1 hour at −30° C. 2-(Trimethylsilyl)ethoxymethyl chloride (8.2 mL, 46.41 mmol, 3 eq) was then added dropwise and the cold bath was removed. The reaction mixture was stirred at ambient temperature for 12 hours and the solvent was removed by rotary evaporation. The residue was dissolved in ethyl acetate and washed with water and brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure to give the desired compound as yellow oil used as crude for the next reaction. ¹H NMR (400 MHz, CDCl₃) δ 7.34 (s, 1H), 7.33 (s, 1H), 7.20 (s, 1H), 7.15 (s, 1H), 5.49 (dd, J=10.0, 1.8 Hz, 2H), 4.64 (dd, J=9.9, 7.5 Hz, 2H), 4.56 (dt, J=8.9, 5.7 Hz, 2H), 4.21 (dt, J=8.6, 4.4 Hz, 2H), 4.09-3.93 (m, 4H), 3.91 (s, 3H), 3.82-3.61 (m, 6H), 3.61-3.50 (m, 2H), 2.90-2.76 (m, 2H), 2.03-1.97 (m, 2H), 1.97-1.86 (m, 4H), 1.75-1.64 (m, 2H), 1.34-1.19 (m, 3H), 1.10 (d, J=2.7 Hz, 9H), 1.08 (d, J=2.7 Hz, 9H), 0.96 (ddd, J=9.1, 6.9, 2.0 Hz, 4H), 0.86 (d, J=2.9 Hz, 9H), 0.85 (d, J=2.9 Hz, 9H), 0.09 (s, 6H), 0.07 (s, 6H), 0.01 (d, J=3.2 Hz, 18H).

(c) (S)-7-hydroxy-8-((5-(((S)-7-methoxy-2-methyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (I18)

(i) (2R,11aS)-2-hydroxy-8-((5-(((2R,11aS)-2-hydroxy-5,11-dioxo-7-((triisopropylsilyl)oxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-10-((2-(trimethylsilyl)ethoxy)methyl)-2,3-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (I14)

Crude I13 (15.74 mmol) was dissolved in dry tetrahydrofuran (40 mL) and a solution of 1% v/v conc. HCl in methanol (120 mL). The reaction mixture was stirred 2 hours at ambient temperature; diluted with ethyl acetate and washed with water (two times), saturated aqueous sodium bicarbonate and brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure to give the desired compound as yellow oil used as crude for the next reaction. LC/MS 2.08 min, (ES+) m/z (relative intensity) 1027.40 [M+H]⁺, ¹H NMR (400 MHz, CDCl₃) δ 7.32 (s, 1H), 7.32 (s, 1H), 7.20 (s, 1H), 7.14 (s, 1H), 5.49 (dd, J=10.0, 3.2 Hz, 2H), 4.69-4.57 (m, 3H), 4.33-4.24 (m, 2H), 4.16-3.93 (m, 4H), 3.88 (s, 3H), 3.89-3.55 (m, 6H), 3.00-2.88 (m, 2H), 2.53 (br, 1H), 2.17-2.05 (m, 2H), 2.00-1.86 (m, 4H), 1.78-1.63 (m, 3H), 1.46-1.39 (m, 2H), 1.36-1.19 (m, 4H), 1.09 (s, 9H), 1.07 (s, 9H), 1.01-0.92 (m, 4H), 0.02 (s, 18H).

(ii) (S)-7-methoxy-10-((2-(trimethyl)-8-((5-(((S)-2,5,11-trioxo-7-((triisopropylsilyl)oxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-2,3,5,10,11,11a-hexahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy) pentyl)oxy)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-2,5,11(3H,10H,11aH)-trione (I15)

TCCA (1.62 g, 7.00 mmol, 1.2 eq) was added to a solution of crude I14 (5.84 mmol), TEMPO (90 mg, 0.58 mmol, 0.1 eq) and sodium acetate (1.14 g, 14.00 mmol, 2.4 eq) in dichloromethane (120 mL) at −10° C. (acetone/ice). The reaction was stirred for 30 min and filtered through celite. The reaction mixture was then quenched with saturated aqueous sodium bicarbonate. The organic phase washed with sodium thiosulfate and brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 20% to 70% ethyl acetate in hexane). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product (3.21 g, 54% over 3 steps). LC/MS 2.17 min, (ES+) m/z (relative intensity) 1023.75 [M+H]⁺, ¹H NMR (400 MHz, CDCl₃) δ 7.32 (s, 2H), 7.23 (s, 1H), 7.18 (s, 1H), 5.53 (dd, J=10.0, 1.6 Hz, 2H), 4.70 (t, J=9.6 Hz, 2H), 4.66-4.58 (m, 2H), 4.22 (d, J=20.1 Hz, 2H), 4.13-3.95 (m, 4H), 3.91 (s, 3H), 3.89-3.74 (m, 4H), 3.72-3.62 (m, 2H), 3.56 (ddd, J=19.2, 5.8, 3.1 Hz, 2H), 2.78 (dd, J=19.3, 9.9 Hz, 2H), 1.95 (dd, J=14.6, 7.3 Hz, 4H), 1.76-1.63 (m, 2H), 1.33-1.23 (m, 3H), 1.10 (s, 9H), 1.09 (s, 9H), 1.02-0.90 (m, 4H), 0.02 (s, 18H).

(iii) (S)-8-((5-(((S)-5,11-dioxo-2-(((trifluoromethyl) sulfonyl)oxy)-7-((triisopropylsilyl)oxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-7-methoxy-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-2-yl trifluoromethanesulfonate (I16)

Anhydrous 2,6-lutidine (2.12 mL, 18.17 mmol, 6.2 eq) was injected in one portion to a solution of I15 (3 g, 2.93 mmol) in dry dichloromethane (40 mL) at −50° C. (acetone/dry ice). Triflic anhydride (2.12 mL, 18.17 mmol, 6 eq) was then added slowly whilst monitoring the temperature. After 20 minutes, the reaction, following a mini work-up (dichloromethane/water), was revealed to be complete by LC/MS. Cold water was added to the still cold reaction mixture and the organic layer was separated and washed with cold saturated sodium bicarbonate, brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 5% to 20% ethyl acetate in hexane). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the product (3.1 g, 82%). LC/MS 2.37 min, no ionisation, ¹H NMR (400 MHz, CDCl₃) δ 7.32 (s, 2H), 7.23 (s, 1H), 7.19 (s, 1H), 7.14 (s, 1H), 7.11 (s, 1H), 5.54 (d, J=10.0 Hz, 2H), 4.74-4.66 (m, 2H), 4.63 (d, J=11.0 Hz, 2H), 4.09-3.96 (m, 4H), 3.94-3.87 (m, 2H), 3.90 (s, 3H), 3.82-3.76 (m, 2H), 3.69-3.65 (m, 2H), 3.22-3.08 (m, 2H), 1.99-1.90 (m, 4H), 1.75-1.64 (m, 2H), 1.33-1.21 (m, 3H), 1.11 (d, J=1.1 Hz, 9H), 1.09 (d, J=1.1 Hz, 9H), 1.01-0.91 (m, 4H), 0.02 (s, 18H).

(iv) (S)-7-methoxy-2-methyl-8-((5-(((S)-2-methyl-5,11-dioxo-7-((triisopropylsilyl)oxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (I17)

Triphenylarsine (588 mg, 1.92 mmol, 0.8 eq) was added to a mixture of triflate I16 (3.1 g, 2.4 mmol, 1 eq), methylboronic acid (1.0 g, 16.8 mmol, 7 eq), silver oxide (4.45 g, 19.2 mmol, 8 eq) and potassium phosphate tribasic (6.1 g, 28.8 mmol, 12 eq) in dry dioxane (40 mL) under an argon atmosphere. The reaction was flushed with argon 3 times and bis(benzonitrile)palladium(II) chloride (184 mg, 0.48 mmol, 0.2 eq) was added. The reaction was flushed with argon 3 more times before being stirred at 70° C. After 1 hour the reaction was observed to be complete by TLC and filtered through a pad celite. The solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to column flash chromatography (silica gel; 20% to 50% ethyl acetate/hexane). Pure fractions were collected and combined, and excess eluent was removed by rotary evaporation under reduced pressure afforded the product (1.1 g, 36%). LC/MS 2.34 min, no ionisation, ¹H NMR (400 MHz, CDCl₃) δ 7.35 (s, 2H), 7.20 (s, 1H), 7.16 (s, 1H), 6.68 (d, J=1.3 Hz, 1H), 6.64 (d, J=1.4 Hz, 1H), 5.53 (s, 1H), 5.51 (s, 1H), 4.67 (t, J=10.1 Hz, 2H), 4.45 (dt, J=10.5, 3.2 Hz, 2H), 4.08-3.94 (m, 4H), 3.90 (s, 3H), 3.77 (dd, J=8.9, 7.5 Hz, 2H), 3.68 (dt, J=10.0, 5.2 Hz, 2H), 3.43 (d, J=16.5 Hz, 2H), 2.78 (d, J=10.4 Hz, 2H), 2.00-1.86 (m, 4H), 1.83 (s, 3H), 1.82 (s, 3H), 1.72-1.68 (m, 2H), 1.30-1.25 (m, 3H), 1.09 (s, 9H), 1.06 (s, 9H), 0.99-0.94 (m, 4H), 0.02 (s, 18H).

(v) (S)-7-hydroxy-8-((5-(((S)-7-methoxy-2-methyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-10-((2-(trimethylsilyl)ethoxy)methyl)-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepine-5,11(10H,11aH)-dione (I18)

Lithium acetate (110 mg, 1.08 mmol, 1 eq) was added to a solution of compound I17 (1.1 g, 1.08 mmol) in wet dimethylformamide (20 mL, 50:1 DMF/water). The reaction was stirred for 12 hours at ambient temperature and diluted with ethyl acetate and washed with citric acid (pH ˜3), brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure to provide the product (1.03 g, quantitative).

LC/MS 1.98 min, (ES+) m/z (relative intensity) 863.35 [M+H]⁺, ¹H NMR (400 MHz, CDCl₃) δ 7.44 (s, 1H), 7.35 (s, 1H), 7.20 (s, 1H), 7.19 (s, 1H), 6.70-6.62 (m, 2H), 6.21 (br, 1H), 5.51 (dd, J=10.0, 5.3 Hz, 2H), 4.66 (dd, J=11.3, 10.1 Hz, 2H), 4.46 (dd, J=10.4, 3.2 Hz, 2H), 4.18-3.99 (m, 4H), 3.90 (s, 3H), 3.78 (td, J=9.7, 6.9 Hz, 2H), 3.67 (td, J=9.7, 6.8 Hz, 2H), 3.49-3.35 (m, 2H), 2.81-2.68 (m, 2H), 2.00-1.89 (m, 4H), 1.82 (s, 3H), 1.81 (s, 3H), 1.75-1.63 (m, 2H), 0.97 (ddd, J=9.9, 6.6, 3.3 Hz, 4H), 0.01 (s, 18H).

(d) Allyl ((S)-1-(((S)-1-((4-((((3-bromopropyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (I21)

Triethylamine (0.35 mL, 2.51 mmol, 2.2 eq) was added to a stirred solution of the 3-bromopropylamine hydrobromide 120 (250 mg, 1.14 mmol) and triphosgene (122 mg, 0.41 mmol, 0.36 eq) in dry tetrahydrofuran (5 mL). The progress of the isocyanate reaction was monitored by periodically removing aliquots from the reaction mixture and quenching with methanol and performing LC/MS analysis. Once the isocyanate formation was complete a solution of the alloc-Val-Ala-PABOH 119 (650 mg, 1.71 mmol, 1.5 eq) and triethylamine (0.24 mL, 1.71 mmol, 1.5 eq) in dry tetrahydrofuran (5 mL) was rapidly added by injection to the freshly prepared isocyanate. The reaction mixture was allowed to stir at 40° C. for 2 hours. As the starting materials were not soluble, 1 mL of DMF was added and the reaction was stirred at 40° C. for 12 hours when it showed around 40% conversion. Excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; gradient, 50% ethyl acetate to 100% ethyl acetate in hexane). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the desired product (190 mg, 31%). LC/MS 1.48 min (no ionisation). ¹H NMR (400 MHz, DMSO) δ 9.97 (s, 1H), 8.14 (d, J=7.0 Hz, 1H), 7.57 (d, J=8.5 Hz, 2H), 7.28 (d, J=8.5 Hz, 2H), 7.23 (d, J=8.6 Hz, 1H), 5.99-5.82 (m, 1H), 5.30 (d, J=17.2 Hz, 1H), 5.17 (d, J=10.2 Hz, 1H), 4.94 (s, 2H), 4.52-4.45 (m, 2H), 4.43-4.40 (m, 1H), 3.93-3.83 (m, 1H), 3.51 (t, J=6.6 Hz, 2H), 3.17 (d, J=5.0 Hz, 1H), 3.11 (dd, J=12.6, 6.6 Hz, 2H), 1.99-1.90 (m, 3H), 1.30 (d, J=7.1 Hz, 3H), 0.89 (t, J=7.1 Hz, 3H), 0.84 (d, J=6.7 Hz, 3H).

Example 1

(a) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl(3-(((S)-8-((5-(((S)-7-methoxy-2-methyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5,11-dioxo-10-((2-(trimethylsilyl)ethoxy)methyl)-5,10,11,11a-tetrahydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)propyl) carbamate (1)

Tetrabutylammonium iodide (16 mg, 0.042 mmol, 0.2 eq), potassium carbonate (43 mg, 0.314 mmol, 1.5 eq) and 121 (170 mg, 0.314 mmol, 1.5 eq) were added to a solution of alcohol I18 (180 mg, 0.209 mmol) in dimethylformamide (3 mL). The reaction mixture was stirred for 2 hour at 80° C. after when the reaction was observed to be complete by LC/MS. The reaction mixture was diluted with ethyl acetate and washed with water three times, brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; gradient, 50% ethyl acetate to 100% ethyl acetate in hexane). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the desired product (180 mg, 65%). LC/MS 2.01 min, (ES+) m/z (relative intensity) 1323.45 [M+H]⁺, ¹H NMR (400 MHz, CDCl₃) δ 8.64 (s, 1H), 7.47 (d, J=8.1 Hz, 2H), 7.31 (s, 2H), 7.24-7.23 (m, 2H), 7.17 (s, 2H), 6.71-6.64 (m, 2H), 6.62 (d, J=7.5 Hz, 1H), 5.93-5.83 (m, 1H), 5.49 (dd, J=10.0, 2.2 Hz, 2H), 5.30-5.26 (m, 2H), 5.28 (d, J=17.0 Hz, 1H), 5.00 (s, 2H), 4.74-4.65 (m, 2H), 4.60-4.55 (m, 2H), 4.49-4.38 (m, 2H), 3.99 (br, 6H), 3.82 (s, 3H), 3.79-3.72 (m, 2H), 3.70-3.57 (m, 2H), 3.44-3.40 (m, 4H), 2.74 (dd, J=16.6, 10.8 Hz, 2H), 2.01 (br, 4H), 1.85-1.82 (m, 10H), 1.60 (br, 2H), 1.42 (d, J=6.8 Hz, 3H), 1.01-0.85 (m, 10H), 0.03-0.09 (m, 18H).

(b) 4-((S)-2-((S)-2-(((allyloxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl (3-(((S)-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)propyl)carbamate (2)

Compound 1 (180 mg, 0.136 mmol) was dissolved in dry THF (5 mL) and cooled to −78° C. Lithium triethylborohydride (0.34 mL, 0.34 mmol, 2.5 eq) was then added dropwise. The reaction was stirred under argon at −78° C. After 30 minutes, the reaction, following a mini work-up (water), was revealed to be complete by LC/MS. The cold bath was removed and water added. The reaction mixture was extracted with ethyl acetate and washed with brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was dissolved with a mixture of dichloromethane/methanol/water (3/6/1, 2 mL/6 mL/0.7 mL). Silica gel was added until the solution gets thick and left stirring at ambient temperature for 5 days. The reaction mixture was filtered and washed with brine, dried over magnesium sulphate; filtered and excess solvent was removed by rotary evaporation under reduced pressure. The resulting residue was subjected to column flash chromatography (silica gel; 5% methanol/chloroform). Pure fractions were collected and combined, and excess eluent was removed by rotary evaporation under reduced pressure afforded the desired product (82 mg, 58%). LC/MS 1.49 min, (ES+) m/z (relative intensity) 1031.45 [M+H]⁺, ¹H NMR (400 MHz, CDCl₃) δ 8.73 (s, 1H), 7.87 (s, 1H), 7.81 (d, J=4.0 Hz, 1H), 7.48 (br, 4H), 7.28-2.26 (m, 2H), 7.17 (d, J=5.5 Hz, 1H), 6.80-6.75 (m, 4H), 5.92-5.89 (m, 1H), 5.78 (br, 1H), 5.30 (d, J=8.5 Hz, 2H), 5.22 (d, J=10.8 Hz, 1H), 5.02 (s, 2H), 4.61-4.57 (m, 4H), 4.27-4.24 (m, 2H), 4.05-3.76 (m, 6H), 3.87 (s, 3H), 3.79-3.70 (m, 2H), 3.49-3.37 (m, 4H), 3.22-3.10 (m, 1H), 2.98-2.94 (m, 1H), 2.20-2.08 (m, 1H), 2.02 (br, 4H), 1.84 (s, 3H), 1.79 (s, 3H), 1.41 (d, J=6.8 Hz, 3H), 1.02-0.82 (m, 6H).

(c) 4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl (3-(((S)-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)propyl)carbamate (3)

Tetrakis(triphenylphosphine)palladium(0) (6 mg, 0.005 mmol, 0.06 eq) was added to a solution of compound 2 (80 mg, 0.077 mmol) and pyrrolidine (16 μL, 0.193 mmol, 2.5 eq) in dry dichloromethane (2 mL). The reaction was flushed with argon three times and stirred 30 minutes at room temperature. Then the reaction was diluted with dichloromethane and washed sequentially with saturated aqueous ammonium chloride and brine. The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resulting residue was used as a crude mixture for the next reaction. LC/MS 1.15 min, (ES+) m/z (relative intensity) 947.55 [M+H]⁺.

(d) 4-((2S,5S)-37-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-2-methyl-4,7,35-trioxo-10,13,16,19,22,25,28,31-octaoxa-3,6,34-triazaheptatriacontanamido)benzyl (3-(((S)-8-((5-(((S)-7-methoxy-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-8-yl)oxy)pentyl)oxy)-2-methyl-5-oxo-5,11a-dihydro-1H-benzo[e]pyrrolo[1,2-a][1,4]diazepin-7-yl)oxy)propyl)carbamate (4)

1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDCl, 15 mg, 0.080 mmol, 1.1 eq) was added to a solution of crude 3 (0.077 mmol) and Mal-(PEG)₈-acid (48 mg, 0.080 mmol, 1.1 eq) in dry dichloromethane (2 mL). The reaction was degassed three times with Argon and stirred for 2 hours and the presence of starting material was no longer observed by LC/MS. The reaction was diluted with dichloromethane and washed sequentially with water and brine. The organic phase was dried over magnesium sulphate filtered and excess dichloromethane removed by rotary evaporation under reduced pressure. The resulting residue was subjected to flash column chromatography (silica gel; 100% chloroform to 10% methanol in chloroform). Pure fractions were collected and combined and excess eluent was removed by rotary evaporation under reduced pressure to give the desired product (40 mg). This residue was then purified further by preparative HPLC (3.7 mg, 31% over 2 steps). LC/MS 1.40 min, (ES+) m/z (relative intensity) 1521.95 [M+H]⁺. ¹H NMR (400 MHz, CDCl₃) δ 8.72 (s, 1H), 7.83 (s, 1H), 7.80 (d, J=3.9 Hz, 1H), 7.63-7.61 (m, 2H), 7.47 (d, J=8.3 Hz, 2H), 7.26 (br, 2H), 6.77-6.75 (m, 4H), 6.69 (s, 2H), 5.02 (s, 2H), 4.64-4.62 (m, 2H), 4.24-4.21 (m, 4H), 4.19-3.95 (m, 4H), 3.89 (s, 2H), 3.86-3.75 (m, 4H), 3.63 (br, 32H), 3.53-3.33 (m, 8H), 3.18-3.14 (m, 2H), 2.98-2.94 (m, 2H), 2.53-5.49 (m, 3H), 2.03 (s, 2H), 1.83 (br, 10H), 1.56 (br, 2H), 1.42 (s, 3H), 1.05-0.90 (m, 6H).

Example 2

General Antibody Conjugation Procedure

Antibodies are diluted to 1-5 mg/mL in a reduction buffer (examples: phosphate buffered saline PBS, histidine buffer, sodium borate buffer, TRIS buffer). A freshly prepared solution of TCEP (tris(2-carboxyethyl)phosphine hydrochloride) is added to selectively reduce cysteine disulfide bridges. The amount of TCEP is proportional to the target level of reduction, within 1 to 4 molar equivalents per antibody, generating 2 to 8 reactive thiols. After reduction for several hours at 37° C., the mixture is cooled down to room temperature and excess drug-linker can be added as a diluted DMSO solution (final DMSO content of up to 10% volume/volume of reaction mixture). The mixture is gently shaken at either 4° C. or room temperature for the appropriate time, generally 1-3 hours. Excess reactive thiols can be reacted with a ‘thiol capping reagent’ like N-ethyl maleimide (NEM) at the end of the conjugation. Antibody-drug conjugates may be concentrated using centrifugal spin-filters with a molecular weight cut-off of 10 kDa or higher, then purified by tangential flow filtration (TFF) or Fast Protein Liquid Chromatography (FPLC). Corresponding antibody-drug conjugates may be determined by analysis by High-Performance Liquid Chromatography (HPLC) or Ultra-High-Performance Liquid Chromatography (UHPLC) to assess drug-per-antibody ratio (DAR) using reverse-phase chromatography (RP) or Hydrophobic-Interaction Chromatography (HIC), coupled with UV-Visible, Fluorescence or Mass-Spectrometer detection; aggregate level and monomer purity can be analysed by HPLC or UHPLC using size-exclusion chromatography coupled with UV-Visible, Fluorescence or Mass-Spectrometer detection. Final conjugate concentration can be determined by a combination of spectroscopic (absorbance at 280, 214 and 330 nm) and biochemical assay (bicinchonic acid assay BCA; Smith, P. K., et al. (1985) Anal. Biochem. 150 (1): 76-85; using a known-concentration IgG antibody as reference). Antibody-drug conjugates are generally sterile filtered using 0.2 μm filters under aseptic conditions, and stored at +4° C., −20° C. or −80° C.

Conjugate A (Herceptin-4, ConjA)

Herceptin™ (3.5 mg, 23.3 nanomoles) was diluted into 3.15 mL of a reduction buffer containing 10 mM sodium borate pH 8.4, 2.5 mM EDTA and a final antibody concentration of 1.11 mg/mL. A 10 mM solution of TCEP was added (1.65 molar equivalent/antibody, 38.5 nanomoles, 3.85 μL) and the reduction mixture was heated at +37° C. for 1.5 hours in an incubator. After cooling down to room temperature, compound 4 was added as a DMSO solution (25 molar equivalent/antibody, 583 nanomoles, in 0.35 mL DMSO). The solution was mixed for 1.5 hours at room temperature, then the conjugation was quenched by addition of N-acetyl cysteine (1.6 micromole, 116.7 μL at 10 mM), then injected into an AKTA™ Pure FPLC using a GE Healthcare HiLoad™ 26/600 column packed with Superdex 200 PG, eluting with 2.6 mL/min of sterile-filtered phosphate-buffered saline (PBS). Fractions corresponding to ConjA monomer peak were pooled, concentrated using a 15 mL Amicon Ultracell 50 KDa MWCO spin filter, analysed and sterile-filtered.

UHPLC analysis on a Shimadzu Prominence system using a Phenomenex Aeris 3.6u XB-C18 150×2.1 mm column eluting with a gradient of water and acetonitrile on a reduced sample of ConjA at 280 nm and 330 nm (Compound 4 specific) shows a mixture of light and heavy chains attached to several molecules of compound 4, consistent with a drug-per-antibody ratio (DAR) of 2.14 molecules of compound 4 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh Bioscience TSKgel G3000SWXL 5 μm 7.8×300 mm column (with a 7 μm 6.0×40 mm guard column) eluting with sterile-filtered SEC buffer containing 200 mM potassium phosphate pH 6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample of ConjA at 280 nm shows a monomer purity of over 99% with no impurity detected. UHPLC SEC analysis gives a concentration of final ConjA at 0.77 mg/mL in 3.2 mL, obtained mass of ConjA is 2.47 mg (71% yield).

Example 3: In Vivo ADC Efficacy Studies

CB.17 SCID mice, aged 8-12 weeks, may be injected with 1 mm³ tumour fragments sub cutaneously in the flank. When tumours reach an average size of 100-150 mg, treatment may be begun. Mice may be weighed twice a week. Tumour size may be measured twice a week. Animals may be monitored individually. The endpoint of the experiment is a tumour volume of 1000 mm³ or 60 days, whichever comes first. Responders can be followed longer.

Groups of 10 xenografted mice can be injected i.v. with 0.2 ml of antibody drug conjugate (ADC), or naked antibody, in phosphate buffered saline (vehicle) or with 0.2 ml of vehicle alone. The concentration of ADC can be adjusted to give, for example, 0.3 or 1.0 mg ADC/kg body weight in a single dose. Three identical doses may be given to each mouse at intervals of, for example, 1 week.

Example 4: In Vitro ADC Efficacy Studies

Medium from subconfluent (about 80-90% confluency) SK-BR-3 cells in a T75 flask was aspirated and PBS (about 20 ml) was added to rinse away the culture medium. The PBS was aspirated and Trypsin-EDTA (5 ml) added. The flask was returned to the 37° C. gassed incubator for up to about 5 minutes. The flask was rapped sharply to dislodge and dissociate cells from the plastic. The cell suspension was transferred to a sterile 50 ml screw-top centrifuge tube. Medium (McCoy's+10% FCS) was added to a final volume of 15 ml, then the tube was centrifuged (400 g for 5 min). The supernatant was aspirated and the pellet re-suspended in 10 ml culture medium. Repeated aspiration (up and down a 10 ml pipette) may be necessary to break up cell clumps and produce monodisperse cell suspensions suitable for counting. Cell suspension (10 μl) was mixed with Trypan blue (10 μl) and live/dead cells counted with a haemocytometer to determine cell concentration and viability. The cell suspension was diluted to 20×10⁴/ml and 50 μl was dispensed into clear 96 well flat bottomed plates. The cells were incubated overnight to allow recovery before use.

A stock solution (1 ml) of antibody drug conjugate (ADC) (20 μg/ml) was made by dilution of filter-sterilised ADC into cell culture medium. A set of 8×10-fold dilutions of stock ADC was made in a 24 well plate by serial transfer of 100 μl onto 900 μl of cell culture medium. 50 μl of each ADC dilution is dispensed into 4 replicate wells of the 96 well plate, containing 50 μl cell suspension seeded the previous day. Control wells receive 50 μl cell culture medium. The 96-well plate containing cells and ADCs was incubated at 37° C. in a CO₂-gassed incubator for 4 days. At the end of the incubation period, viable cells were measured by MTS assay. MTS (Promega) was dispensed (20 μl per well) into each well and incubated for 4 hours at 37° C. in the CO₂-gassed incubator. Well absorbance was measured at 490 nm. Percentage cell survival is calculated from the mean absorbance in the 4 ADC-treated wells compared to the mean absorbance in the 4 control wells (100%).

ADC EC₅₀ (μg/ml) ConjA 0.00257

Abbreviations

-   Ac acetyl -   Acm acetamidomethyl -   Alloc allyloxycarbonyl -   Boc di-tert-butyl dicarbonate -   t-Bu tert-butyl -   Bzl benzyl, where Bzl-OMe is methoxybenzyl and Bzl-Me is     methylbenzene -   Cbz or Z benzyloxy-carbonyl, where Z—Cl and Z—Br are chloro- and     bromobenzyloxy carbonyl respectively -   DMF N,N-dimethylformamide -   Dnp dinitrophenyl -   DTT dithiothreitol -   Fmoc 9H-fluoren-9-ylmethoxycarbonyl -   imp N-10 imine protecting group:     3-(2-methoxyethoxy)propanoate-Val-Ala-PAB -   MC-OSu maleimidocaproyl-O—N-succinimide -   Moc methoxycarbonyl -   MP maleimidopropanamide -   Mtr 4-methoxy-2,3,6-trimethtylbenzenesulfonyl -   PAB para-aminobenzyloxycarbonyl -   PEG ethyleneoxy -   PNZ p-nitrobenzyl carbamate -   Psec 2-(phenylsulfonyl)ethoxycarbonyl -   TBDMS tert-butyldimethylsilyl -   TBDPS tert-butyldiphenylsilyl -   Teoc 2-(trimethylsilyl)ethoxycarbonyl -   Tos tosyl -   Troc 2,2,2-trichlorethoxycarbonyl chloride -   Trt trityl -   Xan xanthyl

REFERENCES

The following references are incorporated by reference in their entirety:

-   EP 0522868 -   EP 0875569 -   EP 1295944 -   EP 1347046 -   EP 1394274 -   EP 1394274 -   EP 1439393 -   JP 05003790 -   JP 2004113151 -   JP 58180487 -   US 2001/055751 -   US 2002/034749 -   US 2002/042366 -   US 2002/150573 -   US 2002/193567 -   US 2003/0228319 -   US 2003/060612 -   US 2003/064397 -   US 2003/065143 -   US 2003/091580 -   US 2003/096961 -   US 2003/105292 -   US 2003/109676 -   US 2003/118592 -   US 2003/119121 -   US 2003/119122 -   US 2003/119125 -   US 2003/119126 -   US 2003/119128 -   US 2003/119129 -   US 2003/119130 -   US 2003/119131 -   US 2003/124140 -   US 2003/124579 -   US 2003/129192 -   US 2003/134790-A1 -   US 2003/143557 -   US 2003/157089 -   US 2003/165504 -   US 2003/185830 -   US 2003/186372 -   US 2003/186373 -   US 2003/194704 -   US 2003/206918 -   US 2003/219806 -   US 2003/224411 -   US 2003/224454 -   US 2003/232056 -   US 2003/232350 -   US 20030096743 -   US 20030130189 -   US 2003096743 -   US 2003130189 -   US 2004/0001827 -   US 2004/005320 -   US 2004/005538 -   US 2004/005563 -   US 2004/005598 -   US 2004/0101899 -   US 2004/018553 -   US 2004/022727 -   US 2004/044179 -   US 2004/044180 -   US 2004/101874 -   US 2004/197325 -   US 2004/249130 -   US 20040018194 -   US 20040052793 -   US 20040052793 -   US 20040121940 -   US 2005/271615 -   US 2006/116422 -   U.S. Pat. No. 4,816,567 -   U.S. Pat. No. 5,362,852 -   U.S. Pat. No. 5,440,021 -   U.S. Pat. No. 5,583,024 -   U.S. Pat. No. 5,621,002 -   U.S. Pat. No. 5,644,033 -   U.S. Pat. No. 5,674,713 -   U.S. Pat. No. 5,700,670 -   U.S. Pat. No. 5,773,223 -   U.S. Pat. No. 5,792,616 -   U.S. Pat. No. 5,854,399 -   U.S. Pat. No. 5,869,445 -   U.S. Pat. No. 5,976,551 -   U.S. Pat. No. 6,011,146 -   U.S. Pat. No. 6,153,408 -   U.S. Pat. No. 6,214,345 -   U.S. Pat. No. 6,218,519 -   U.S. Pat. No. 6,268,488 -   U.S. Pat. No. 6,518,404 -   U.S. Pat. No. 6,534,482 -   U.S. Pat. No. 6,555,339 -   U.S. Pat. No. 6,602,677 -   U.S. Pat. No. 6,677,435 -   U.S. Pat. No. 6,759,509 -   U.S. Pat. No. 6,835,807 -   U.S. Pat. No. 7,223,837 -   U.S. Pat. No. 7,375,078 -   U.S. Pat. No. 7,521,541 -   U.S. Pat. No. 7,723,485 -   WO 00/012508 -   WO 00/12507 -   WO 00/12508 -   WO 01/16318 -   WO 01/45746 -   WO 02/088172 -   WO 03/026577 -   WO 03/043583 -   WO 04/032828 -   WO 2000/12130 -   WO 2000/14228 -   WO 2000/20579 -   WO 2000/22129 -   WO 2000/32752 -   WO 2000/36107 -   WO 2000/40614 -   WO 2000/44899 -   WO 2000/55351 -   WO 2000/75655 -   WO 200053216 -   WO 2001/00244 -   WO 2001/38490 -   WO 2001/40269 -   WO 2001/40309 -   WO 2001/41787 -   WO 2001/46232 -   WO 2001/46261 -   WO 2001/48204 -   WO 2001/53463 -   WO 2001/57188 -   WO 2001/62794 -   WO 2001/66689 -   WO 2001/72830 -   WO 2001/72962 -   WO 2001/75177 -   WO 2001/77172 -   WO 2001/88133 -   WO 2001/90304 -   WO 2001/94641 -   WO 2001/98351 -   WO 2002/02587 -   WO 2002/02624 -   WO 2002/06317 -   WO 2002/06339 -   WO 2002/101075 -   WO 2002/10187 -   WO 2002/102235 -   WO 2002/10382 -   WO 2002/12341 -   WO 2002/13847 -   WO 2002/14503 -   WO 2002/16413 -   WO 2002/16429 -   WO 2002/22153 -   WO 2002/22636 -   WO 2002/22660 -   WO 2002/22808 -   WO 2002/24909 -   WO 2002/26822 -   WO 2002/30268 -   WO 2002/38766 -   WO 2002/54940 -   WO 2002/59377 -   WO 2002/60317 -   WO 2002/61087; -   WO 2002/64798 -   WO 2002/71928 -   WO 2002/72596 -   WO 2002/78524 -   WO 2002/81646 -   WO 2002/83866 -   WO 2002/86443 -   WO 2002/88170 -   WO 2002/89747 -   WO 2002/92836 -   WO 2002/94852 -   WO 2002/98358 -   WO 2002/99074 -   WO 2002/99122 -   WO 2003/000842 -   WO 2003/002717 -   WO 2003/003906 -   WO 2003/003984 -   WO 2003/004989 -   WO 2003/008537 -   WO 2003/009814 -   WO 2003/014294 -   WO 2003/016475 -   WO 2003/016494 -   WO 2003/018621 -   WO 2003/022995 -   WO 2003/023013 -   WO 2003/024392 -   WO 2003/025138 -   WO 2003/025148 -   WO 2003/025228 -   WO 2003/026493 -   WO 2003/029262 -   WO 2003/029277 -   WO 2003/029421 -   WO 2003/034984 -   WO 2003/035846 -   WO 2003/042661 -   WO 2003/045422 -   WO 2003/048202 -   WO 2003/054152 -   WO 2003/055439 -   WO 2003/055443 -   WO 2003/062401 -   WO 2003/062401 -   WO 2003/072035 -   WO 2003/072036 -   WO 2003/077836 -   WO 2003/081210 -   WO 2003/083041 -   WO 2003/083047 -   WO 2003/083074 -   WO 2003/087306 -   WO 2003/087768 -   WO 2003/088808 -   WO 2003/089624 -   WO 2003/089904 -   WO 2003/093444 -   WO 2003/097803 -   WO 2003/101283 -   WO 2003/101400 -   WO 2003/104270 -   WO 2003/104275 -   WO 2003/105758 -   WO 2003004529 -   WO 2003042661 -   WO 2003104399 -   WO 2004/000997 -   WO 2004/001004 -   WO 2004/009622 -   WO 2004/011611 -   WO 2004/015426 -   WO 2004/016225 -   WO 2004/020595 -   WO 2004/022709 -   WO 2004/022778 -   WO 2004/027049 -   WO 2004/031238 -   WO 2004/032828 -   WO 2004/032842 -   WO 2004/040000 -   WO 2004/043361 -   WO 2004/043963 -   WO 2004/044178 -   WO 2004/045516 -   WO 2004/045520 -   WO 2004/045553 -   WO 2004/046342 -   WO 2004/047749 -   WO 2004/048938 -   WO 2004/053079 -   WO 2004/063355 -   WO 2004/063362 -   WO 2004/063709 -   WO 2004/065577 -   WO 2004/074320 -   WO 2004000221 -   WO 2004020583 -   WO 2004042346 -   WO 2004065576 -   WO 2005/023814 -   WO 2005/082023 -   WO 2005/085251 -   WO 2006/111759 -   WO 2007/044515 -   WO 2007/085930 -   WO 2009/052249 -   WO 2010/091150 -   WO 91/02536 -   WO 92/07574 -   WO 92/17497 -   WO 94/10312 -   WO 94/28931 -   WO 9630514 -   WO 97/07198 -   WO 97/44452 -   WO 98/13059 -   WO 98/37193 -   WO 98/40403 -   WO 98/51805 -   WO 98/51824 -   WO 99/28468 -   WO 99/46284 -   WO 99/58658 -   Am. J. Hum. Genet. 49 (3):555-565 (1991) -   Amiel J., et al Hum. Mol. Genet. 5, 355-357, 1996 -   Amir et al (2003) Angew. Chem. Int. Ed. 42:4494-4499 -   Amsberry, et al (1990) J. Org. Chem. 55:5867 -   Angew Chem. Intl. Ed. Engl. (1994) 33:183-186 -   Annu. Rev. Neurosci. 21:309-345 (1998) -   Arai H., et al J. Biol. Chem. 268, 3463-3470, 1993 -   Arai H., et al Jpn. Circ. J. 56, 1303-1307, 1992 -   Arima, et al., J. Antibiotics, 25, 437-444 (1972) -   Attie T., et al, Hum. Mol. Genet. 4, 2407-2409, 1995 -   Auricchio A., et al Hum. Mol. Genet. 5:351-354, 1996 -   Barel M., et al Mol. Immunol. 35, 1025-1031, 1998 -   Barella et al (1995) Biochem. J. 309:773-779 -   Barnett T., et al Genomics 3, 59-66, 1988 -   Beck et al (1992) J. Mol. Biol. 228:433-441 -   Beck et al (1996) J. Mol. Biol. 255:1-13 -   Berge, et al., J. Pharm. Sci., 66, 1-19 (1977) -   Biochem. Biophys. Res. Commun. (2000) 275(3):783-788 -   Biochem. Biophys. Res. Commun. 255 (2), 283-288 (1999) -   Blood (2002) 100 (9):3068-3076 -   Blood 99 (8):2662-2669 (2002) -   Blumberg H., et al Cell 104, 9-19, 2001 -   Bose, et al., Tetrahedron, 48, 751-758 (1992) -   Bourgeois C., et al J. Clin. Endocrinol. Metab. 82, 3116-3123, 1997 -   Brinster et al (1988) Proc. Natl. Acad. Sci. USA 85:836 -   Buchman and Berg (1988) Mol. Cell. Biol. 8:4395 -   Cancer Res. 61 (15), 5857-5860 (2001) -   Carl et al (1981) J. Med. Chem. 24:479-480 -   Carlsson et al (1978) Biochem. J. 173:723-737 -   Carter, P. (2006) Nature Reviews Immunology 6:343-357 -   Cell 109 (3):397-407 (2002) -   CellTiter Glo Luminescent Cell Viability Assay, Promega Corp.     Technical Bulletin TB288 -   Chakravarty et al (1983) J. Med. Chem. 26:638-644 -   Chan, J. and Watt, V. M., Oncogene 6 (6), 1057-1061 (1991) -   Child et al (1999) J. Biol. Chem. 274: 24335-24341 -   Cho H.-S., et al Nature 421, 756-760, 2003 -   Ciccodicola, A., et al EMBO J. 8(7):1987-1991 (1989) -   Clackson et al (1991) Nature, 352:624-628 -   Clark H. F., et al Genome Res. 13, 2265-2270, 2003 -   Corey E, Quinn J E, Buhler K R, et al. LuCap35: a new model of     prostate cancer progression to androgen independence. The Prostate     2003; 55:239-46 -   Coussens L., et al Science (1985) 230(4730):1132-1139 -   Cree et al (1995) AntiCancer Drugs 6:398-404 -   Crouch et al (1993) J. Immunol. Meth. 160:81-88 -   Davis et al (2001) Proc. Natl. Acad. Sci USA 98(17):9772-9777 -   de Groot et al (2001) J. Org. Chem. 66:8815-8830 -   de Groot et al (2003) Angew. Chem. Int. Ed. 42:4490-4494 -   Dennis et al. (2002) “Albumin Binding As A General Strategy For     Improving The Pharmacokinetics Of Proteins” J Biol Chem.     277:35035-35043 -   Dobner et al (1992) Eur. J. Immunol. 22:2795-2799 -   Dornan et al (2009) Blood 114(13):2721-2729 -   Doronina et al (2006) Bioconj. Chem. 17:114-124 -   Dubowchik et al. Bioconjugate Chemistry, 2002, 13, 855-869 -   Dubowchik, et al. (1997) Tetrahedron Letters, 38:5257-60 -   Dumoutier L., et al J. Immunol. 167, 3545-3549, 2001 -   E. Schröder and K. Lübke, The Peptides, volume 1, pp 76-136 (1965)     Academic Press -   Ehsani A., et al (1993) Genomics 15, 426-429 -   Eliel, E. and Wilen, S., “Stereochemistry of Organic Compounds”,     John Wiley & Sons, Inc., New York, 1994 -   Elshourbagy N. A., et al J. Biol. Chem. 268, 3873-3879, 1993 -   Erickson et al (2006) Cancer Res. 66(8):1-8 -   Feild, J. A., et al (1999) Biochem. Biophys. Res. Commun. 258     (3):578-582 -   Fields, G. and Noble, R. (1990) “Solid phase peptide synthesis     utilizing 9-fluoroenylmethoxycarbonyl amino acids”, Int. J. Peptide     Protein Res. 35:161-214 -   Fuchs S., et al Mol. Med. 7, 115-124, 2001 -   Fujisaku et al (1989) J. Biol. Chem. 264 (4):2118-2125) -   Gary S. C., et al Gene 256, 139-147, 2000 -   Gaugitsch, H. W., et al (1992) J. Biol. Chem. 267 (16):11267-11273) -   Geiser et al “Automation of solid-phase peptide synthesis” in     Macromolecular Sequencing and Synthesis, Alan R. Liss, Inc., 1988,     pp. 199-218 -   Genome Res. 13 (10):2265-2270 (2003) -   Genomics 62 (2):281-284 (1999) -   Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146 -   Getz et al (1999) Anal. Biochem. Vol 273:73-80 -   Glynne-Jones et al (2001) Int J Cancer. October 15; 94(2):178-84 -   Gregson et al., Chem. Commun. 1999, 797-798 -   Gregson et al., J. Med. Chem. 2001, 44, 1161-1174 -   Gu Z., et al Oncogene 19, 1288-1296, 2000 -   Ha et al (1992) J. Immunol. 148(5):1526-1531 -   Haendler B., et al J. Cardiovasc. Pharmacol. 20, s1-S4, 1992 -   Hamann P. (2005) Expert Opin. Ther. Patents 15(9):1087-1103 -   Hamblett et al (2004) Clin. Cancer Res. 10:7063-7070 -   Handbook of Pharmaceutical Additives, 2nd Edition (eds. M. Ash     and I. Ash), 2001 (Synapse Information Resources, Inc., Endicott,     N.Y., USA) -   Handbook of Pharmaceutical Excipients, 2nd edition, 1994 -   Hara, et al., J. Antibiotics, 41, 702-704 (1988) -   Hashimoto et al (1994) Immunogenetics 40(4):287-295 -   Hay et al. (1999) Bioorg. Med. Chem. Lett. 9:2237 -   Herdwijn, P. et al., Canadian Journal of Chemistry. 1982, 60, 2903-7 -   Hermanson, G. T. (1996) Bioconjugate Techniques; Academic Press: New     York, p 234-242 -   Hochlowski, et al., J. Antibiotics, 40, 145-148 (1987) -   Hofstra R. M. W., et al Eur. J. Hum. Genet. 5, 180-185, 1997 -   Hofstra R. M. W., et al Nat. Genet. 12, 445-447, 1996 -   Horie et al (2000) Genomics 67:146-152 -   Hubert, R. S., et al (1999) Proc. Natl. Acad. Sci. U.S.A. 96     (25):14523-14528) -   Hurley and Needham-VanDevanter, Acc. Chem. Res., 19, 230-237 (1986) -   Immunogenetics 54 (2):87-95 (2002) -   Int. Rev. Cytol. 196:177-244 (2000) -   Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988) -   J. Biol. Chem. 270 (37):21984-21990 (1995) -   J. Biol. Chem. 276 (29):27371-27375 (2001) -   J. Biol. Chem. 277 (22):19665-19672 (2002) -   J. Biol. Chem. 278 (33):30813-30820 (2003) -   Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno     Biology, 5th Ed., Garland Publishing, New York -   Jeffrey et al (2005) J. Med. Chem. 48:1344-1358 -   Jonsson et al (1989) Immunogenetics 29(6):411-413 -   Junutula, et al., 2008b Nature Biotech., 26(8):925-932 -   Kang, G-D., et al., Chem. Commun., 2003, 1680-1689 -   Kasahara et al (1989) Immunogenetics 30(1):66-68 -   King et al (2002) Tetrahedron Letters 43:1987-1990 -   Kingsbury et al (1984) J. Med. Chem. 27:1447 -   Kohler et al (1975) Nature 256:495 -   Kohn, in Antibiotics Ill. Springer-Verlag, New York, pp. 3-11     (1975). -   Konishi, et al., J. Antibiotics, 37, 200-206 (1984) -   Kovtun et al (2006) Cancer Res. 66(6):3214-3121 -   Kuhns J. J., et al J. Biol. Chem. 274, 36422-36427, 1999 -   Kuminoto, et al., J. Antibiotics, 33, 665-667 (1980) -   Kurebayashi et al (1999) Brit. Jour. Cancer 79(5-6):707-717 -   Lab. Invest. 82 (11):1573-1582 (2002) -   Lambert J. (2005) Current Opin. in Pharmacol. 5:543-549 -   Langley and Thurston, J. Org. Chem., 52, 91-97 (1987) -   Larhammar et al (1985) J. Biol. Chem. 260(26):14111-14119 -   Law et al (2006) Cancer Res. 66(4):2328-2337 -   Le et al (1997) FEBS Lett. 418(1-2):195-199 -   Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988) -   Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965) -   Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965) -   Levenson et al (1997) Cancer Res. 57(15):3071-3078 -   Liang et al (2000) Cancer Res. 60:4907-12 -   Manfre, F. et al., J. Org. Chem. 1992, 57, 2060-2065 -   Marks et al (1991) J. Mol. Biol., 222:581-597 -   McDonagh (2006) Protein Eng. Design & Sel., 19(7): 299-307 -   Mendoza et al (2002) Cancer Res. 62:5485-5488 -   Miller et al (2003) Jour. of Immunology 170:4854-4861 -   Miura et al (1996) Genomics 38(3):299-304 -   Miura et al (1998) Blood 92:2815-2822 -   Moore M., et al Proc. Natl. Acad. Sci. U.S.A. 84, 9194-9198, 1987 -   Morrison et al (1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855 -   Muller et al (1992) Eur. J. Immunol. 22 (6):1621-1625 -   Mungall A. J., et al Nature 425, 805-811, 2003 -   Nagase T., et al (2000) DNA Res. 7 (2):143-150) -   Nakamuta M., et al Biochem. Biophys. Res. Commun. 177, 34-39, 1991 -   Nakayama et al (2000) Biochem. Biophys. Res. Commun. 277(1):124-127 -   Naruse et al (2002) Tissue Antigens 59:512-519 -   Nature 395 (6699):288-291 (1998) -   Neuberger and Williams (1988) Nucleic Acids Res. 16:6713 -   Novabiochem Catalog 2006/2007 -   Ogawa Y., et al Biochem. Biophys. Res. Commun. 178, 248-255, 1991 -   Okamoto Y., et al Biol. Chem. 272, 21589-21596, 1997 -   Oncogene 10 (5):897-905 (1995) -   Oncogene 14(11):1377-1382 (1997)) -   Parrish-Novak J., et al J. Biol. Chem. 277, 47517-47523, 2002 -   Payne, G. (2003) Cancer Cell 3:207-212 -   Phillips et al (2008) Cancer Res. 68(22):9280-9290 -   Pingault V., et al (2002) Hum. Genet. 111, 198-206 -   Pletnev S., et al (2003) Biochemistry 42:12617-12624 -   Preud'homme et al (1992) Clin. Exp. Immunol. 90(1):141-146 -   Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (7):4126-4131 -   Proc. Natl. Acad. Sci. U.S.A. 93 (1):136-140 (1996) -   Proc. Natl. Acad. Sci. U.S.A. 98 (17):9772-9777 (2001) -   Proc. Natl. Acad. Sci. U.S.A. 99 (26):16899-16903 (2002) -   Proc. Natl. Acad. Sci. U.S.A. 96 (20):11531-11536 (1999) -   Protective Groups in Organic Synthesis, Greene and Wuts, 3^(rd)     Edition, 1999, John Wiley & Sons Inc. -   Puffenberger E. G., et al Cell 79, 1257-1266, 1994 -   Rao et al (1997) Breast Cancer Res. and Treatment 45:149-158 -   Reiter R. E., et al Proc. Natl. Acad. Sci. U.S.A. 95, 1735-1740,     1998 -   Remington's Pharmaceutical Sciences, 20th edition, pub. Lippincott,     Williams & Wilkins, 2000 -   Rodrigues et al (1995) Chemistry Biology 2:223 -   Ross et al (2002) Cancer Res. 62:2546-2553 -   S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)     McGraw-Hill Book Company, New York -   Sakaguchi et al (1988) EMBO J. 7(11):3457-3464 -   Sakamoto A., Yanagisawa M., et al Biochem. Biophys. Res. Commun.     178, 656-663, 1991 -   Sanderson et al (2005) Clin. Cancer Res. 11:843-852 -   Semba K., et al Proc. Natl. Acad. Sci. U.S.A. 82, 6497-6501, 1985 -   Servenius et al (1987) J. Biol. Chem. 262:8759-8766 -   Shamis et al (2004) J. Am. Chem. Soc. 126:1726-1731 -   Sheikh F., et al (2004) J. Immunol. 172, 2006-2010 -   Shimizu, et al, J. Antibiotics, 29, 2492-2503 (1982) -   Sinha S. K., et al (1993) J. Immunol. 150, 5311-5320 -   Storm et al (1972) J. Amer. Chem. Soc. 94:5815 -   Strausberg et al (2002) Proc. Natl. Acad. Sci USA 99:16899-16903 -   Sun et al (2002) Bioorganic & Medicinal Chemistry Letters     12:2213-2215 -   Sun et al (2003) Bioorganic & Medicinal Chemistry 11:1761-1768 -   Svensson P. J., et al Hum. Genet. 103, 145-148, 1998 -   Swiercz J. M., et al J. Cell Biol. 165, 869-880, 2004 -   Syrigos and Epenetos (1999) Anticancer Research 19:605-614 -   Takeuchi, et al., J. Antibiotics, 29, 93-96 (1976) -   Tawaragi Y., et al Biochem. Biophys. Res. Commun. 150, 89-96, 1988 -   ten Dijke, P., et al Science 264 (5155):101-104 (1994) -   Thompson, J. S., et al Science 293 (5537), 2108-2111 (2001) WO     2004/058309 -   Thurston, et al., Chem. Brit., 26, 767-772 (1990) -   Thurston, et al., Chem. Rev. 1994, 433-465 (1994) -   Toki et al (2002) J. Org. Chem. 67:1866-1872 -   Tonnelle et al (1985) EMBO J. 4(11):2839-2847 -   Touchman et al (2000) Genome Res. 10:165-173 -   Trail et al (2003) Cancer Immunol. Immunother. 52:328-337 -   Tsunakawa, et al., J. Antibiotics, 41, 1366-1373 (1988) -   Tsutsumi M., et al Gene 228, 43-49, 1999 -   Uchida et al (1999) Biochem. Biophys. Res. Commun. 266:593-602 -   Verheij J. B., et al Am. J. Med. Genet. 108, 223-225, 2002 -   Von Hoegen et al (1990) J. Immunol. 144(12):4870-4877 -   Webster et al (1994) Semin. Cancer Biol. 5:69-76 -   Weis J. J., et al J. Exp. Med. 167, 1047-1066, 1988 -   Weis J. J., et al Proc. Natl. Acad. Sci. U.S.A. 83, 5639-5643, 1986 -   Wilson et al (1991) J. Exp. Med. 173:137-146 -   Wu et al (2005) Nature Biotech. 23(9):1137-1145 -   Xie et al (2006) Expert. Opin. Biol. Ther. 6(3):281-291 -   Xu, M. J., et al (2001) Biochem. Biophys. Res. Commun. 280     (3):768-775 WO 2004/016225 -   Xu, X. Z., et al Proc. Natl. Acad. Sci. U.S.A. 98 (19):10692-10697     (2001) -   Yamaguchi, N., et al Biol. Chem. 269 (2), 805-808 (1994) -   Yamamoto T., et al Nature 319, 230-234, 1986 -   Yu et al (1992) J. Immunol. 148(2) 633-637 

The invention claimed is:
 1. A conjugate of formula (A):

and salts and solvates thereof, wherein: D represents either group D1 or D2:

the dotted line indicates the optional presence of a double bond between C2 and C3; when there is a double bond present between C2 and C3, R² is selected from the group consisting of: (ia) C₅₋₁₀ aryl group, optionally substituted by one or more substituents selected from the group comprising: halo, nitro, cyano, ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃ alkylene; (ib) C₁₋₅ saturated aliphatic alkyl; (ic) C₃₋₆ saturated cycloalkyl; (id)

wherein each of R³¹, R³² and R³³ are independently selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where the total number of carbon atoms in the R² group is no more than 5; (ie)

wherein one of R^(35a) and R^(35b) is H and the other is selected from: phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; and (if)

where R³⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃ alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted by a group selected from halo, methyl, methoxy; pyridyl; and thiophenyl; (ig) halo; when there is a single bond present between C2 and C3, R² is

where R^(36a) and R^(36b) are independently selected from H, F, C₁₋₄ saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups are optionally substituted by a group selected from C₁₋₄ alkyl amido and C₁₋₄ alkyl ester; or, when one of R^(16a) and R^(16b) is H, the other is selected from nitrile and a C₁₋₄ alkyl ester; R⁶ and R⁹ are independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, NO₂, SnMe₃ and halo; either (a) R¹⁰ is H, and R¹¹ is OH or OR^(A), where R^(A) is C₁₋₄ alkyl; or (b) R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound; or (c) R¹⁰ is H and R¹¹ is OSO_(Z)M, where z is 2 or 3 and M is a monovalent pharmaceutically acceptable cation; or (d) R¹¹ is OH or OR^(A), where R^(A) is C₁₋₄ alkyl and R¹⁰ is selected from:

where R^(z) is selected from: (z-i)

(z-ii) OC(═O)CH₃; (z-iii) NO₂; (z-iv) OMe; (z-v) glucoronide; (z-vi) —C(═O)—X₁—NHC(═O)X₂—NH—R^(ZC), where —C(═O)—X₁—NH— and —C(═O)—X₂—NH— represent natural amino acid residues and R^(zc) is selected from Me, OMe, OCH₂CH₂OMe; Y is selected from formulae A1 and A2:

Z¹ is a C₁₋₃ alkylene group; Z² is a C₁₋₃ alkylene group; Q is:

where Q^(X) is such that Q is an amino-acid residue, a dipeptide residue or a tripeptide residue; L is a linker connected to a cell binding agent; CBA is the cell binding agent; n is an integer between 0 and 48; R and R′ are each independently selected from optionally substituted C₁₋₁₂ alkyl, C₃₋₂₀ heterocyclyl and C₅₋₂₀ aryl groups, and optionally in relation to the group NRR′, R and R′ together with the nitrogen atom to which they are attached form an optionally substituted 4-, 5-, 6- or 7-membered heterocyclic ring; R⁸ is either: (a) independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, NO₂, SnMe₃ and halo; or (b) of formula A*:

wherein: D′ represents either group D′1 or D2:

wherein the dotted line indicates the optional presence of a double bond between C2′ and C3′; R¹⁷ is independently selected from H, R, OH, OR, SH, SR, NH₂, NHR, NRR′, NO₂, SnMe₃ and halo; R″ is a C₃₋₁₂ alkylene group, which chain may be interrupted by one or more heteroatoms, e.g. O, S, N(H), NMe and/or aromatic rings, e.g. benzene or pyridine, which rings are optionally substituted; and X and X′ are independently selected from O, S and N(H); and R²², R¹⁶, R¹⁹, R²⁰ and R²¹ are as defined for R², R⁶, R⁹, R¹⁰ and R¹¹ respectively.
 2. The conjugate according to claim 1, wherein R⁹ is H and R⁶ is H.
 3. The conjugate according to claim 1, wherein D is D1, there is a double bond between C2 and C3, and R² is: a C₅₋₇ aryl group which bears one to three substituent groups selected from methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholino and methylthiophenyl; or (b) a C₁₋₅ saturated aliphatic alkyl group; or (c) a C₃₋₆ saturated cycloalkyl group; or (d) a group of formula:

wherein the total number of carbon atoms in the R² group is no more than 4; or (e)

or (f) a group of formula:

wherein R³⁴ is selected from H, methyl, ethyl, ethenyl and ethynyl.
 4. The conjugate according to claim 1, wherein D is D1, there is a double bond between C2 and C3, and R² is

and: (a) R^(36a) and R^(36b) are both H; or (b) R^(36a) and R^(36b) are both methyl; or (c) R^(36a) and R^(36b) is H, and the other is selected from C₁₋₄ saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups are optionally substituted.
 5. The conjugate according to claim 1, wherein R¹⁰ is H, and R¹¹ is OH.
 6. The conjugate according to claim 1, wherein R¹⁰ and R¹¹ form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are bound.
 7. The conjugate according to claim 1, wherein R¹¹ is OH or OR^(A) and R¹⁰ is selected from:

wherein —C(═O)—X₁—NHC(═O)X₂—NH—, selected from: -Phe-Lys-, -Val-Ala- -Val-Lys-, -Ala-Lys- and -Val-Cit-.
 8. The conjugate according to claim 1, wherein R⁸ is OR^(8A), where R^(8A) is optionally substituted C₁₋₄ alkyl.
 9. The conjugate according to claim 1, wherein R⁸ is of formula A*.
 10. The conjugate according to claim 9, wherein X and X′ are O, R″ is C₃₋₇ alkylene and R¹⁷ is OR^(17A), where R^(17A) is optionally substituted C₁₋₄ alkyl.
 11. The conjugate according to claim 9, wherein R¹⁶, R¹⁹, R²⁰, R²¹ and D′ are the same as R⁶, R⁹, R¹⁰, R¹¹ and D respectively.
 12. The conjugate according to claim 1, wherein L is of formula: -L^(A)-(CH₂)_(m)- ehere m is from 0 to 6; and L^(A) is selected from:

where Ar represents a C₅₋₆ arylene group.
 13. The conjugate according to claim 1, wherein Q is selected from: ^(NH)-Phe-Lys-^(C═O), ^(NH)Val-Ala-^(C═O), ^(NH)Val-Lys-^(C═O), ^(NH)Ala-Lys-^(C═O), ^(NH)-Val-Cit-^(C═O), ^(NH)-Phe-Cit-^(C═O), ^(NH)-Leu-Cit-^(C═O), ^(NH)-Ile-Cit-^(C═O), ^(NH)-Phe-Arg-^(C═O), and ^(NH)-Trp-Cit-^(C═O); where Cit is citrulline.
 14. A pharmaceutical composition comprising the conjugate of claim 1 a pharmaceutically acceptable diluent, carrier or excipient.
 15. A compound of formula (B):

wherein: Y^(L) is selected from formulae B1 and B2:

G is a linker for connecting to a cell binding agent; and D, R⁶, R⁸, R⁹, R¹⁰, R¹¹, Z¹ and Z², Q and n are as defined in claim
 1. 16. The compound according to claim 15, wherein G is of formula: G^(A)-(CH₂)_(m)- where m is from 0 to 6; and G^(A) is selected from:

where Ar represents a C₅₋₆ arylene group.
 17. A compound of formula (C):

wherein Y^(c) is selected from formulae C1 and C2:

and D, R⁶, R⁸, R⁹, R¹⁰, R¹¹, Z¹ and Z² are as defined in claim
 1. 